Medicament for use in a method of inducing or extending a cellular cytotoxic immune response

ABSTRACT

The present invention relates to a medicament for use in a method of inducing a cellular cytotoxic immune response, the method comprising the steps of: i) administering to a patient a delivery system comprising (a) a molecule binding to a receptor on the surface of a dendritic cell, (b) an antigen-comprising protein bound to molecule of (a) and (c) a first adjuvant, wherein upon binding of the molecule of (a) to the receptor, the protein of (b) is internalized and processed in the dendritic cell and the antigen comprised in the protein is presented on the surface of the dendritic cell, thereby activating a T cell in the patient; and ii) administering to the patient a re-activator selected from the group consisting of (d) complexed interleukin 2 (IL-2cx), (e) a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant, and (f) a combination of (d) and (e), wherein the peptide is derived from the antigen-comprising protein as defined in step i), thereby reactivating the T cell activated in step i), wherein the re-activator of step ii) is administered in a time frame of from 0 h to 14 days after the administration of the delivery system of step i).

The present invention relates to a medicament for use in a method of inducing a cellular cytotoxic immune response, the method comprising the steps of:

-   -   i) administering to a patient a delivery system comprising (a) a         molecule binding to a receptor on the surface of a dendritic         cell, (b) an antigen-comprising protein bound to molecule of (a)         and (c) a first adjuvant, wherein upon binding of the molecule         of (a) to the receptor, the protein of (b) is internalized and         processed in the dendritic cell and the antigen comprised in the         protein is presented on the surface of the dendritic cell,         thereby activating a T cell in the patient; and     -   ii) administering to the patient a re-activator selected from         the group consisting of (d) complexed interleukin 2         (IL-2cx), (e) a peptide-loaded major histocompatibility complex         class I (MHC-I) presenting cell and a second adjuvant, and (f) a         combination of (d) and (e), wherein the peptide is derived from         the antigen-comprising protein as defined in step i), thereby         re-activating the T cell activated in step i),

wherein the re-activator of step ii) is administered in a time frame of from 0 h to 14 days after the administration of the delivery system of step i).

The immune system protects the body against pathogens and tumor cells by a variety of mechanisms. To function properly, it has to discriminate between “self” and “foreign” (pathogens/tumors). It detects and fights a variety of pathogens, including bacteria, viruses, parasites, fungi, and toxins. The immune systems of vertebrates such as humans consist of many types of proteins, cells, tissues, and organs, which interact in a dynamic network. As part of this complex immune response, the vertebrate immune system adapts over time to recognize particular pathogens more efficiently. The adaptation process creates immunological memory and allows a more effective protection during future encounters with these pathogens. Vaccination is based on this process of acquired immunity.

Dendritic cells (DCs) form part of the immune system. Their main function is to process antigen material and present it on their surface to other cells of the immune system, thus functioning as antigen-presenting cells.

T helper cells (also known as effector T cells or T_(h) cells) are also an important member of the immune system in that they play a fundamental role in establishing and maximizing the capabilities of the immune system. T_(h) cells are involved in activating and directing other immune cells. They are essential in determining B cell antibody class switching, in the activation and expansion of cytotoxic T cells, and in maximizing bactericidal activity of phagocytes such as macrophages. It is this diversity in function and their role in influencing other cells that gives T helper cells their name. Proliferating helper T cells that develop into effector T cells differentiate into two major subtypes of cells known as T_(h)1 and T_(h)2 cells (also known as Type 1 and Type 2 helper T cells, respectively), wherein T_(h)2 cells mainly promote the humoral immune system (stimulation of B cells into proliferation, induction of B cell antibody class switching, and increase of antibody production), whereas T_(h)1 cells promote mainly the cellular immune system (maximization of killing efficacy of the macrophages and cytotoxic CD8⁺ T cells). Depending on the nature of the invading pathogen, the immune system develops a Th1 or Th2 immune response. In the case of the Th1 immune response, the CD8⁺ T cells show a strong tendency for differentiation into cytotoxic T cells. At the same time, both the CD8⁺ and CD4⁺ helper T cells of the Th1 immune response secrete large amounts of IFN-γ (and other Th1 cytokines/chemokines) and elicit the generation of antibodies predominantly of the IgG2a and IgG2b isotype in the mouse and predominantly of the IgG isotype in the human. The Th1 immune response is particularly effective for defending the body against viruses, (intracellular) bacteria and tumors.

Currently available vaccines and adjuvant systems directed against live, attenuated, or inactivated pathogenic components mainly elicit an antibody immune response, but not an effective Th1 cytotoxic response (Steinman et al., 2007, Nature 449, 419-26). The induced antibodies bind to components of the pathogen and thus biologically inactivate it (“neutralizing antibodies”). However, there are a number of diseases, where neutralizing antibodies are not sufficient to protect from the disease or to control the disease and current vaccine technology is not effective. These are diseases which may require an effective Th1 immune response for containment and/or eradication of the infection. Examples are tuberculosis, malaria, leishmania, prion diseases, orthomyxoviruses and in particular influenza, hepatitis A, hepatitis B, human immunodeficiency virus (HIV) and other lentiviruses, cytomegalovirus, herpesviruses, papillomaviruses, bunyaviruses, caliciviruses, filoviruses, flaviviruses and in particular hepatitis C virus, papillomaviruses, paramyxoviruses, a variety of respiratory viruses, and other viruses which need for containment and eradication an effective Th1 immune response, and in particular a Th1 cytotoxic response. The development of a vaccination methodology inducing such an effective Th1 response is therefore highly desirable. Furthermore, targeting the mechanism of “cross-presentation” (see below) is of paramount importance for the induction of a Th1 immune response against viral, bacterial, parasitic, and fungal pathogens, since dendritic cells most often do not become directly infected in the course of an infection. Without the development of a Th1 immune response, many viral, bacterial, parasitic, or fungal infections cannot be contained or eradicated in the human body.

It has been found in WO 2009/065561, that cells playing a major role in the induction of the Th1 response can be selectively targeted. It was found that chemokine (C motif) receptor 1 (XCR1) is present on the surface of professional antigen-presenting cell, particularly dendritic cells (DC), and can be used to selectively deliver substance into these cells. Targeted delivery of a substance to XCR1-bearing DC allows induction of a potent Th1 immune reaction in mammals/humans. Current vaccines mainly address the Th2 antigen presentation pathway and mainly lead to the generation of Th2-type (neutralizing) antibodies and immune reactions. In particular, through targeting to XCR1-bearing DC, a Th1-type humoral and cellular (cytotoxic) immune reaction can be elicited to a given immunogen. It can be anticipated that NK cells, CD8⁺ T cells, and Th1CD4⁺ T cells participate in this reaction, but other CD4⁺ T cells may also contribute to this type of reaction. An adjuvant, either alone or in combination with an immunogen or any pharmaceutical compound, can be selectively targeted to XCR1-bearing antigen-presenting cells (APC).

In the periphery, the immune system has to discriminate between harmless foreign or self-antigens on the one hand and dangerous (viral, bacterial, fungal, parasitic, toxin-like) antigens on the other hand. The antigen is taken up by the DC and broken down to peptides (“processed”). The resultant peptides are “presented” to T lymphocytes (T cells) in the context of the MHC class I or MHC class II. The CD4⁺ subset of T cells recognizes the antigen in the context of MHC class II, the CD8⁺ subset of T cells recognizes the antigen in the context of MHC class I. Concomitant with the uptake of antigen, the DC is capable of sensing through a large set of “danger signal” recognition receptors (e.g. toll-like receptors, NOD-like receptors), whether the antigen is of dangerous nature or whether it is harmless. The patterns recognized by the “danger signal” recognition receptors (also designated “pattern recognition receptors”) are usually molecular structures that are unique to microorganisms. These can be cell wall components (e.g. lipopolysaccharide, peptidoglycan) or nucleic acid modifications (e.g. unmethylated CpG motifs) in case of microbes, or structural features and modifications that are unique to viral DNA or viral RNA (e.g. double-stranded

RNA). Also cells dying from apoptosis in the body release molecules which are capable of triggering “danger signal” recognition receptors (e.g. High Mobility Group Protein B1, heat-shock proteins).

In the case of a dangerous antigen, the DC activates a specific response program (“maturation”). The antigen is presented to CD4⁺ and CD8⁺ T cells, which simultaneously receive from the DC additional signals indicating the dangerous nature of the antigen. As a result, both T cell subsets become activated, expand extensively with a prolonged life span and develop to “effector T cells”. These can be CD4⁺ T cells providing “help” to other DC or B cells or other cells of the immune system, or can be even CD4⁺ cytotoxic cells. Within the CD8⁺ T cell subset, again T helper cells develop, but a large proportion of CD8⁺ T cells become effector cells capable of eliminating the invading pathogen through secretion of IFN-γ and other soluble factors or through killing of infected body cells. As a result of the T cell help to B cells, antigen-specific B cells differentiate to plasma cells which secrete antibodies directed to the antigen (pathogen). These antibodies help to fight the pathogen through a number of mechanisms (e.g. neutralization, improved antigen uptake, opsonization, complement fixation).

A certain number of effector CD4⁺ and CD8⁺ T cells survive the acute phase of an immune response to a pathogen and become long-lived “memory T cells”. Memory T cells and long-lived plasma cells orchestrate upon re-exposure to the same pathogen (antigen) a very fast immune response allowing the immune system to eliminate the pathogen (antigen) very effectively. This enhanced capability of the T-cell and B cell immune response upon re-exposure to the same pathogen is termed “immunity” and the antigens which induce immunity are “immunogenic”.

In summary, the T cell compartment of the immune system contains CD4⁺ T cells and CD8⁺ T cells. In the naïve organism, only a few hundred CD4⁺ or CD8⁺ T cells recognize a given antigen. As long as they have not encountered antigen, T cells are in a naïve state and cannot exert effector functions. Naïve CD4⁺ and CD8⁺ T cells can only be activated by APC by soluble protein or polypeptide antigen, which is taken up by the APC, processed, and “presented” on the cell surface in the context of MHC. The source of primary antigen exposure can be a soluble protein or polypeptide or any type of vaccine, such as attenuated infectious agents, viral or bacterial vectors coding for a desired antigenic protein or peptide, or DNA or RNA expression vectors coding for an antigenic protein or peptide. As a result of primary exposure to antigen in the context of danger signals there will exist within days a population of “primed” CD4⁺ and CD8⁺ T cells.

When protein antigen is targeted into an APC, it will be degraded (“processed”) to peptides and these peptides will be presented on the surface of the APC in the context of MHC-II (classical presentation) and MHC-I (“cross-presentation”). Naïve CD4⁺ T cells recognize their peptide-antigen in the context of MHC-II, naïve CD8⁺ T cells in the context of MHC-I. They become activated, proliferate, and acquire effector functions. If the peptides are presented by the DC in the context of a “danger signal”, the expanded CD8⁺ T cells will differentiate into cytotoxic T cells. Only professional APC are capable of inducing this cytotoxic function in naïve CD8⁺ T cells (“priming”, “primary immunization”). Once the CD8⁺ T cells have acquired their cytotoxic potential, they will be able to kill cells in the body which express the same peptides in the context of MHC-I. Thus they will kill cells infected by a given infectious agent or tumor cells.

Antigens which are taken up by cells capable of antigen presentation are not only presented in the context of MHC-I (“cross-presentation”), but also in the context of MHC-II (“classical presentation”). Therefore, any antigen delivery into antigen-presenting cells, together with a danger signal, will not only activate CD8⁺ T cells, but also CD4⁺ T cells. The CD4⁺ T cells will differentiate into Th1 T cells secreting TNF-α and IFN-γ, and some will also develop into cytotoxic T cells.

Primary CD8⁺ T cell activation can be achieved by providing a soluble antigen, or by direct or indirect targeting of antigen(s) into APC (B cells, macrophages, DC), and in particular into XCR1⁺ DC of a naïve host. Indirect targeting is achieved by providing the antigen in cells which are not APC, but which can hand over the antigen to APC, in particular to XCR1⁺ DC in vivo. When the antigen is applied together with a “danger signal” (e.g. LPS, Poly I:C, CpG, etc.), this will induce primary CD8⁺ T cell cytotoxic immunity.

Similar early T cell activation can be expected by antigenic re-challenge (in the presence of danger signals) of a host, which has been primed with antigen before and whose antigen-specific CD8⁺ T cells and CD4⁺ T cells are in a non-activated memory state.

Similar T cell activation can be expected in hosts which harbor low grade chronic infections which cannot be cleared by the host (e.g. CMV, HCV, HIV).

Antigen can be targeted into APC. This targeting can be mediated by monoclonal antibodies (mAb) or antibody fragments binding to surface structures on APC, or by any ligands binding to receptors on the surface of APC. These ligands can be sugar moieties, chemokines, soluble protein ligands, or any other structures allowing internalization of the antigen into the APC. Antigen taken up by any DC is presented both in the context of MHC-I and MHC-II. Direct or indirect targeting of antigen into dendritic cells (DC) substantially improves cross-presentation over other modes of antigen application. Best cross-presentation and thus priming of CD8⁺ T cells can be achieved by targeting of antigen into XCR1⁺ DC (Bachem et al. 2010, J Exp Med 207, 1273-1281, Bachem et al., 2012, Front Immunol 3, 214; Caminschi et al. 2013, Radford et al. 2013, Kreutz et al. 2013).

After an efficient priming of CD8⁺ T cells in the context of a danger signal (which is also a first adjuvant), these cytotoxic T cells will represent approximately 1% to 5% of the CD8⁺ T cell repertoire and exhibit significant cytotoxic potential. For example, the achieved cytotoxicity may protect the host against a pathogen, from which the antigen was derived, and this level of protection can also be effective for newly developing cancerous tissue (FIG. 2B).

However, when attempting to establish long-term immunity against a given antigen or to provide high levels of cytotoxic protection against imminent infections, or to provide high levels of cytotoxicity against already established tumors, the cytotoxic potential induced by an initial priming of naïve CD8⁺ T cells or be re-activation of CD8⁺ T cells may not suffice. Under these circumstances a further amplification of the activated antigen-specific CD8⁺ T cells is necessary.

After recognizing antigen expressed in the context of MHC-I, CD8⁺ T cells become activated, de novo express or strongly upregulate a variety of cell surface molecules, such as CD69, 4-1BB, ICOS, CD25, CD40L, OX40, and proliferate for up to approximately 8 days in the mouse (the timeframe may be somewhat different in the human). Thereafter, the initially activated CD8⁺ T cells gradually return over approximately 2-3 weeks into a resting “memory” state. At the same time, the expanded antigen-specific T cell population strongly contracts. A proportion of the CD8⁺ T cells will survive this process of contraction and will become memory T cells (Cui et al., 2010, Immunol. Rev. 236, 151-166).

Classical vaccination schemes can be divided into an initial priming step, followed by one or several “boostings”. The principle of boosting is based on de novo activation of initially expanded B cells or T cells which have undergone a downmodulation of the initial activation or have already reverted into the resting state.

The problem underlying the present invention is the provision of an improved medicament for use in a method for inducing a cellular cytotoxic immune response and/or in a method for extending a cellular cytotoxic immune response compared to prior art technologies.

The problem is solved by the present invention.

In one embodiment, the present invention relates to a medicament for use in a method of inducing a cellular cytotoxic immune response, the method comprising the steps of:

-   -   i) administering to a patient a delivery system comprising (a) a         molecule binding to a receptor on the surface of a dendritic         cell, (b) an antigen-comprising protein bound to molecule of (a)         and (c) a first adjuvant, wherein upon binding of the molecule         of (a) to the receptor, the protein of (b) is internalized and         processed in the dendritic cell and the antigen comprised in the         protein is presented on the surface of the dendritic cell,         thereby activating a T cell in the patient; and     -   ii) administering to the patient a re-activator selected from         the group consisting of (d) complexed interleukin 2         (IL-2cx), (e) a peptide-loaded major histocompatibility complex         class I (MHC-I) presenting cell and a second adjuvant, and (f) a         combination of (d) and (e), wherein the peptide is derived from         the antigen-comprising protein as defined in step i), thereby         re-activating the T cell activated in step i),

wherein the re-activator of step ii) is administered in a time frame of from 0 h to 14 days after the administration of the delivery system of step i).

In a preferred embodiment, the medicament is for use in a human.

The patient is preferably a mammalian patient, more preferably a human patient. The patient may be suffering from an infection or tumor, or, in case of prophylactic treatment, is not suffering from an infection or tumor, but is to be protected from a respective infection or tumor disease.

In a further preferred embodiment, the receptor on the surface of a dendritic cell is a human receptor on the surface of a dendritic cell.

One possibility to provide strong T cell cytotoxicity within a short time would be to interfere with the down regulatory mechanism becoming effective within days of the initial activation or re-activation of CD8⁺ T cells by antigen. To prevent this down modulation of the CD8⁺ T cell response, we reasoned that we have to provide another stimulus involving the T cell receptor complex on CD8 T cells and/or appropriate growth factors within a short timeframe of the initial activation or re-activation. This concept runs against the current dogma which teaches that repeated activation of T cells via the T cell receptor within a short time will result in “activation-induced cell death” (Gorak-Stolinska et al., 2001, J Leukoc Biol 70, 756-766).

To test our concept, C57BL/6 mice were immunized (“primed”) by targeting an antigen into XCR1⁺ DC in the presence of a first adjuvant which supports a Th-1 response (poly I:C, LPS, CpG, or equivalent). As antigen for this primary immunization we used the model antigen ovalbumin (OVA), or a peptide sequence derived from ovalbumin (SIINFEKL (SEQ ID NO: 11)). This peptide sequence is known to be preferentially presented in the context of the MHC-I of C57BL/6 mice after processing of OVA by the targeted APC. For this initial immunization, the protein OVA or the peptide SIINFEKL (SEQ ID NO: 11) were recombinantly fused either to a monoclonal antibody (mAb) specific for XCR1 or to the chemokine ligand XCL1 binding to XCR1, as described earlier (WO 200/065561). At various time points (3-20 days) after this priming step, mice were re-exposed to antigen. We did not inject protein or peptides into the host, since we anticipated that either procedure would lead to the presentation of antigenic peptides in the context of the MHC-I on host cells, which then would be killed by the already activated antigen-specific CD8⁺ T cells (compare FIG. 1A), a highly undesired effect. Instead, syngeneic splenic cells were isolated from C57BL/6 mice, incubated with the peptide SIINFEKL (SEQ ID NO: 11) in vitro (“loading” of MHC-I), washed, and injected i.v. into mice which have been primed before.

Unexpectedly, and contrary to current knowledge, we could achieve with this regime an amplification of the number of antigen-specific CD8⁺ T cells, when the CD8⁺ T cells were re-exposed to antigen within a narrow timeframe. No amplification was observed, when re-exposure was very early, on day 3, and very limited amplification was observed when re-exposure to antigen was done following day 9 of initial CD8⁺ T cell activation (FIG. 3). Injection of SIINFEKL-loaded syngeneic splenic cells into primed animals alone did not expand the primed CD8⁺ T cell population, only when a second adjuvant (poly I:C, LPS, CpG, or equivalent) was co-applied to provide a “danger” signal, the desired effect was achieved. When the injection of peptide-loaded splenocytes was performed at an optimal time point, the antigen-responsive CD8⁺ T cell population expanded approximately 10-fold, from 0.2 x 10⁶ after priming without amplification, to 2 x 10⁶ cells with amplification (FIG. 4). These expanded CD8⁺ cells expressed high levels of effector molecules such as Granzyme B, perforin, TNF-α and IFN-γ, molecules involved in the CD8⁺ T cell defense of infectious agents or in the eradication of tumors. In the human, the optimal timeframe for the amplification of the initial T cell activation may differ from the optimal timeframe in the mouse (approximately day 5-8 after initial CD8⁺ T cell activation).

When comparing various amplification time points, it became apparent that a re-exposure with antigen 5-8 days after the initial priming gives the highest degree of CD8⁺ T cell expansion and the highest expression of cytotoxic effector molecules (TNF-α, IFN-γ, granzyme B, perforin) in CD8⁺ T cells. Day 5-8 is an early time point following the recognition of antigen by resting CD8⁺ T cells and thus a time point at which the T cells are still strongly activated. Therefore, this amplification does not represent a classical boost system. Instead, this type of amplification provides signals allowing T cells to continue their initial activation and expansion phase instead of entering the usual phase of downregulation and contraction. Because of this particular effect, we have termed this amplification, when applied together with an adjuvant, as “Antigen-Dependent Amplification System” (ADAS).

Therefore, according to the present invention, “Antigen-Dependent Amplification System” or “ADAS” is understood as the second step ii) of the present invention relating to administering to the patient a re-activator, wherein the reactivator is a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant, and wherein the peptide is derived from the antigen-comprising protein as defined in step i) of the present invention, thereby re-activating the T cell activated in step i). Optionally, the reactivator in ADAS further comprises complexed interleukin 2 (IL-2cx).

In order to examine the cellular requirements for the effectiveness of ADAS, various lymphocytic populations (splenocytes, B cells, T cells, and DC) were for comparison loaded with SIINFEKL (SEQ ID NO: 11) in vitro and injected within the optimal timeframe in mice (day 5) together with a second adjuvant (poly I:C, LPS, CpG, or equivalent). This experiment determined that all of these lymphocytic populations expressing MHC-I were capable of providing the signals necessary to continue the initial activation, expansion and functional differentiation of CD8⁺ T cells to cytotoxic effector cells (FIG. 3B). While priming alone resulted on day 10 in 1%-5% of antigen-specific CD8⁺ T cells within the splenic CD8 T cell population, application ADAS raised this frequency to around 15%-25%.

From the results obtained one can conclude that ADAS will work in vivo with any system capable to provide high enough density of peptide-loaded MHC-I molecules on the surface of lymphocytic cells or even non-lymphocytic cells. Instead of loading MHC-I molecules with peptides externally in vitro, one could envisage systems in which cells would be fed in vitro with whole antigen-comprising protein, allowing the cells to process the antigen and present the antigenic peptides in the context of MHC-I. One could also envisage systems, in which cells would be in vitro exposed to viral systems capable of infecting the cells resulting in the expression of high amounts of a peptide in the context of MHC-I. Further, the MHC-I bearing cells could be transfected with expression vectors coding for a given protein or peptide sequence, again resulting in an efficient presentation of peptides in the context of MHC-I.

Since whole antigen delivered to APC will not only be presented in the context of MHC-I to CD8⁺ T cells, but also in the context of MHC-II to CD4⁺ T cells, ADAS can also be used to amplify CD4⁺ T cell responses. For this particular amplification, the cells used for ADAS have to express MHC-II molecules on the cell surface, which would be loaded with appropriate peptides. This loading could be done by external exposure to suitable peptides, or the MHC-II bearing cells could be transfected by expression vectors coding for a given protein or peptide sequence, again resulting in an efficient presentation of peptides in the context of MHC-II.

Although we applied ADAS through i.v. injection, other routes of application of the peptide-loaded cells are possible. This could be done by subcutaneous, intracutaneous, intramuscular injection, intraperitoneal, intrathecal, or by direct injection into tumor tissue.

Therefore, the administration of a reactivator of step ii) of the present invention may be performed by known methods of administration, in particular selected from subcutaneous (s.c.), intracutaneous, i.v., intramuscular injection, intraperitoneal, intrathecal, or by direct injection into tumor tissue, more preferably by i.v. or s.c. injection.

Also, the administration of a delivery system of step i) of the present invention may be performed by known methods of administration, in particular selected from subcutaneous, intracutaneous, i.v., intramuscular injection, intraperitoneal, intrathecal, administration into the lung, or by direct injection into tumor tissue, more preferably by i.v. and s.c. injection.

The molecule binding to a receptor on the surface of a dendritic cell, (b) an antigen-comprising protein bound to molecule of (a) and (c) a first adjuvant are preferably administered as a single pharmaceutical preparation. Such pharmaceutical preparation is preferably a liquid which may further contain pharmaceutical acceptable excipients like buffering compounds. Such pharmaceutical preparation is preferably sterilized.

The volume of the dose of the delivery system of step i) for intramuscular administration is preferably up to about 5 mL, for example, between 0.3 mL and 3 mL, between 1 mL and 3 mL, about 0.5 to 1 mL, or about 2 mL. The amount of active ingredient in each dose should be enough to provide for treatment or prevention. In different embodiments, the unit dose of substance to be delivered should be up to about 5 μg substance/kg body weight, between about 0.2 to 3 μg, between about 0.3 to 1.5 μg, between about 0.4 to 0.8 μg, or about 0.6 μg. In alternative embodiments unit doses could be up to about 6 μg substance/kg body weight, between about 0.05 to 5 μg, or between about 0.1 to 4 μg. Representative amounts of protein per dose are from approximately 1 μg to approximately 1 mg, more preferably from approximately 5 μg to approximately 500 μg, still more preferably from approximately 10 μg to approximately 250 μg and most preferably from approximately 25 μg to approximately 100 μg.

The number of cells of a reactivator comprising a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant to be administered in step ii) can vary and is typically in the range of from 1×10⁶ to 400×10⁶ cells, preferably is in the range of from 1×10⁶ to 200×10⁶ cells.

Complexed IL-2 as reactivator is preferably administered as a solution and/or at a dosage in the range of from 1 μg/kg body weight to 200 μg/kg body weight, more preferably of from 1 μg/kg body weight to 50 μg/kg body weight, even more preferably of from 1 μg/kg body weight to 20 μg/kg body weight wherein the amounts refer to the IL-2 content in the composition.

Although we tested the effect of ADAS after targeting of antigen into XCR1⁺ DC, one can conclude from our results that any system leading to a significant initial activation (“priming”) or re-activation of CD8⁺ T cells and/or CD4⁺ T cells by T cell receptor triggering in vivo can be amplified with the ADAS approach.

The lymphokine IL-2 has been used in the past in order to expand T cell and NK cell populations and was shown to be effective, when provided in a “complexed” from, i.e. bound to an antibody blocking its binding to the high affinity IL-2 receptor chain (CD25, Boyman et al., 2006, Science 311, 1924-1927).

According the present invention, “complexed IL-2” or “IL-2cx” is understood as IL-2 which is non-covalently bound to a binding molecule, in particular antibody or antibody fragment which is blocking its binding to the high affinity IL-2 receptor chain (CD25). In a preferred embodiment, the antibody is humanized or human. In a preferred embodiment, IL-2 is human IL-2. IL-2 may be synthesized synthetically or recombinantly, using an adequate host, more preferably IL-2 is prepared recombinantly.

We tested complexed IL-2 (IL-2cx) in our system of T cell activation through targeting of antigen into XCR1⁺ DC. Contrary to expectations, priming of the T cell response followed by application of IL-2cx alone did not significantly increase the number of antigen-specific CD8⁺ T cells (FIG. 4). Application of ADAS alone on day 5 increased the number of antigen-specific CD8⁺ T cells from around 0.2×10⁶ after priming alone to around 2×10⁶ and thus approximately 10-fold, as described above. However, and very surprisingly, when applied in the context of ADAS, IL-2cx very strongly augmented the amplification of antigen-specific CD8⁺ T cells to approximately 100-200×10⁶ antigen-specific CD8⁺, i.e. approximately by a factor of 50-100. This highly synergistic effect indicated that ADAS created favorable conditions for the biological effects of IL-2, when IL-2 was applied in a complexed form. The combination of ADAS and IL-2cx was amplifying the initial priming to such a degree that now the large majority of all splenic immune cells were composed of antigen-specific CD8⁺ T cells. When further examined, this massively expanded T cell population expressed granzyme B to approximately 60-70% indicating a high cytotoxic potential.

In order to test the cytotoxic capacity of CD8⁺ T cells amplified under various conditions we chose a tumor model, in which a highly aggressive OVA-transfected tumor line is injected s.c. into syngeneic C57BL/6 mice and allowed to grow for 6 days to a substantial size (around 20 mm²). From day 6, groups of mice were either left untreated, or were treated by different regimes. Priming of the tumor-bearing mice on day 6 alone by targeting of OVA into XCR1⁺ DC barely had any effect on the growth of the tumor (FIG. 5) and the same was true if the mice were not primed, but treated with IL-2cx alone. Surprisingly, priming of the mice on day 6, followed by a treatment with IL-2cx alone for the consecutive days significantly reduced the growth of the tumor until day 22, indicating induction of substantial killing capacity in vivo. However, the tumor resumed its growth around day 22 indicating that not all of the tumor mass could be removed. Also the application of ADAS on day 6 after priming very substantially reduced the growth of the tumor which, however, re-started to grow around day 18 (FIG. 5). Interestingly, the application of ADAS on day 6 after priming combined with consecutive application of IL-2cx was clearly most effective in reducing the size of the tumor until the end of the experiment (FIG. 5). The obtained results indicated that injection of IL-2cx alone within a given timeframe after targeting of antigen into APC was already very effective in controlling the growth of the tumor. Further, the outcome indicated that ADAS, when applied within a given timeframe after initial activation of CD8⁺ T cells by antigen, also induced strong killing activity against a tumor expressing this antigen. Most effective in controlling the growth of an aggressive tumor was a combination of ADAS and IL-2cx (FIG. 5). Although in this experiment ADAS was applied first and followed by IL-2cx, the treatment sequence could also be reversed. In this case IL-2cx would be applied first, followed at an appropriate time by ADAS.

In preferred embodiment of step i) of the medicament for use in a method of the present invention is disclosed in WO 2009/065561 for XCR1 as receptor on the surface of a dendritic cell. As disclosed in WO 2009/065561, an anti-XCR1 antibody or fragment thereof, or XCL1 or a functionally active fragment thereof can preferably be employed as molecule binding to a receptor on the surface of a dendritic cell. The disclosure of WO 2009/065561 relating to the delivery system wherein XCR1 is receptor on the surface of a dendritic cell is hereby incorporated by reference and the embodiments disclosed therein also apply for step i) of the medicaments for use of the present invention.

Suitable antigen-comprising proteins are known to the skilled person.

For example, numerous suitable antigen-comprising proteins in the context of tumor diseases are known. Moreover, suitable peptides as vaccines are described, as e.g. summarized in Aranda et al., Oncolmmunology 2:12, e26621; December 2013 for solid neoplasms, including glioma, lung carcinoma, sarcoma, melanoma, esophageal squamous cell carcinoma, gastric cancer, hepatocellular carcinoma, pancreatic cancer, colorectal carcinoma, renal cell carcinoma, prostate cancer, ovarian carcinoma, gynecologic malignancies, and various other tumors.

The antigen-comprising proteins specifically targeted in these clinical trials encompass cancer-testis antigens such as NY-ESO-1, TTK protein kinase (also known as MOS), lymphocyte antigen 6 complex, locus K (LY6K, best known as URLC10), insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3, best known as IMP3), ring finger protein (RNF43), and translocase of outer mitochondrial membrane 34 (TOMM34); carcinoembryonic antigens like glypican-3; differentiation antigens such as melan-A (MLANA) and premelanosome protein (PMEL, best known as gp100); tumor-restricted antigens, such as the SYT-SSX fusion (which is selectively expressed by synovial sarcomas as a result of a t(X;18)(p11;q11) chromosomal translocation); as well as so-called “shared tumor-associated antigens” (antigens that are overexpressed by malignant cells but also produced in normal amounts by one or several healthy tissues), including vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2, survivin, Wilms tumor 1 (WT1), telomerase reverse transcriptase (TERT), and p53. Such proteins are therefore suitable antigen-comprising proteins in the context of tumor. A further summary on peptide vaccines in cancer is Yamada et al., Cancer Sci, 2013, 104(1):15-21.

In case of inducing a cellular cytotoxic immune response in a tumor patient or a patient to be prophylactically treated regarding a tumor, a suitable antigen-comprised protein involved in tumor disease may be used. Such proteins are known in the art. As described e.g. in Tacken and Figdor (Tacken P. J., Figdor C. G.; Targeted antigen delivery and activation of dendritic cells in vivo: Steps towards cost effective vaccines. Semin. Immunol., 2011, 23(1):12-20), ideal tumor antigens are those known as shared tumor-specific antigens because they are selectively expressed in tumor cells, of various histotypes, and not in MHC-expressing normal tissues. Examples of such type of antigens are the MAGE-A or NY-ESO-1 antigens. The various members in this category of antigens are expressed at variable proportions depending on the tumor type and the disease stage. Thus, vaccines based on these antigens require selection of patients bearing tumors that express the target antigen. Another category of tumor antigens that are deemed valuable for vaccine development are those derived from oncogenic proteins which are overexpressed in tumors. Bona fide non-self tumor antigens are derived from two major sources: viral antigens in the case of tumors of oncogenic viral origin such as cervical carcinomas caused by HPV infection, and somatic mutations.

In case of inducing a cellular cytotoxic immune response in patient suffering from an infection or a patient to be prophylactically treated regarding an infectious disease (or infection), suitable antigen-comprised proteins of a pathogen may be used, in particular a pathogen selected from malaria, tuberculosis, leishmania or a virus, in particular a virus selected from an orthomyxovirus, influenza virus, hepatitis A virus, hepatitis B virus, chronic hepatitis C virus, a lentivirus, in particular HI-Virus, cytomegalovirus, a herpes virus, a papillomavirus, a bunyavirus, a calicivirus, a filovirus, a flavivirus, or a respiratory virus. In an even more preferred embodiment, antigen-comprised protein is a protein of a virus selected from a hepatitis C virus, a papillomavirus, a paramyxovirus, or a respiratory virus.

Viruses, in particular RNA viruses, typically exhibit a high mutation rate. However, there are typically conserved regions found in particular in genomic segments encoding non-structural and/or internal proteins, such regions encoding a viral polymerase or a nucleoprotein. Such regions are unsuitable for classical vaccination technologies, as such conserved proteins are not exposed on the viral coat surface. In contrast, such antigen-comprised proteins and/or antigen comprised in the protein may be used in the medicament for use according to the invention.

In a preferred embodiment, the antigen-comprised protein and/or antigen comprised in the protein of a virus is conserved.

In a further preferred embodiment, the antigen-comprised protein is a non-structural protein and/or the antigen is comprised in a non-structural and/or internal protein.

In a yet further preferred embodiment, the antigen-comprised protein is a protein of an RNA virus.

A “non-structural” or “internal” protein is a protein which is not part of the coat or envelope of a virus particle.

In a further embodiment, the antigen is comprised in the protein in step i) is immunodominant. Immunodominant means that, although many pMHC complexes are available for a certain pathogen, T-cell responses are reproducibly focused on one or few key antigens, and such the antigen is one such key antigens.

Suitable antigen-comprised proteins and antigens of influenza A virus are for example described in Wu et al. (PNAS, 2011, 108(22): 9178-9183), and encompass NP (nucleoprotein), basic polymerase 1 and Ml.

Step ii) relates to administering to the patient a re-activator selected from the group consisting of (d) complexed interleukin 2 (IL-2cx), (e) a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant, and (f) a combination of (d) and (e), wherein the peptide is derived from the antigen-comprising protein as defined in step i), thereby re-activating the T cell activated in step i).

In an embodiment, in case of (e), the cell and the second adjuvant are administered in a time frame of from 0 h to 14 days after the administration of the delivery system of step i).

In one preferred embodiment, in case of (e), the cell and the second adjuvant are administered only once in a time frame of from 0 h to 14 days after the administration of the delivery system of step i).

In a preferred embodiment, in case of (e), the cell and the second adjuvant are administered in a time frame of from 48 h to 14 days after the administration of the delivery system of step i).

For example, the cell and the second adjuvant are administered 48 h, 72 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days after the administration of the delivery system of step i).

In a more preferred embodiment, in case of (e), the cell and the second adjuvant are administered in a time frame of from 3 days to 9 days, even more preferably of from 4 days to 8 days, for example 4 days, 5 days, 6 days, 7 days, 8 days after the administration of the delivery system of step i).

In another preferred embodiment, in case of (e), the cell and the second adjuvant are administered only once in a time frame of from 0 h to 14 days, preferably in a time frame of from 48 h to 14 days after the administration of the delivery system of step i).

For example, the cell and the second adjuvant are administered only once 48 h, 72 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days after the administration of the delivery system of step i).

In a further preferred embodiment, in case of (e), the cell and the second adjuvant are administered only once in a time frame of from 3 days to 9 days, even more preferably of from 4 days to 8 days, for example 4 days, 5 days, 6 days, 7 days, 8 days after the administration of the delivery system of step i).

In case of (d), complexed interleukin 2 (IL-2cx) is administered in a time frame of from 0 h to 14 days after the administration of the delivery system of step i).

In case of (d), complexed interleukin 2 (IL-2cx) is preferably administered repeatedly after the administration of the delivery system of step i) in a time frame of from 0 h to 14 days after the administration of the delivery system of step i). It is further possible, that further administration of complexed IL-2 takes place after 14 days after the administration of the delivery system of step i); however, at least one administration is in a time frame of from 0 h to 14 days.

In a further preferred embodiment, in case of (d), complexed interleukin 2 (IL-2cx) is preferably administered in a time frame of from 48 h to 9 days, even more preferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the administration of the delivery system of step i).

In a yet further preferred embodiment, in case of (d), complexed interleukin 2 (IL-2cx) is preferably administered repeatedly in a time frame of from 48 h to 9 days, even more preferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the administration of the delivery system of step i). Thus, complexed interleukin 2 (IL-2cx) is preferably administered 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times in a time frame of from 48 h to 9 days, even more preferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the administration of the delivery system of step i). For example, complexed interleukin 2 (IL-2cx) may be administered daily or every two days. For example, complexed interleukin 2 (IL-2cx) may be administered 48 h, 3 days, 4 days, 5 days, 6 days, 7 days and 8 days after the administration of the delivery system of step i). In another example, complexed interleukin 2 (IL-2cx) may be administered 48 h, 4 days, 6 days, and 8 days after the administration of the delivery system of step i).

It is further possible, that further administration of complexed IL-2 takes place after the indicated time of, e.g. 14 days, preferably 9 or 8 days, after the administration of the delivery system of step i); however, at least one administration is in the indicated time frame.

In case of (f), a combination of complexed interleukin 2 (IL-2cx), and a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant is administered. In a preferred embodiment, the complexed interleukin 2 (IL-2cx) may be administered before, concomitantly or after administration of the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant. Preferably, the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant are only administered once, and the complexed interleukin 2 (IL-2cx) is administered repeatedly in a time frame of from 0 h to 14 days after the administration of the delivery system of step i). In such embodiment, complexed IL-2 may be administered both before, concomitantly and/or after administration of the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant.

For the alternative (f), the same preferred embodiments for administration of complexed IL-2 and peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant, respectively, apply as for alternatives (e) and (f).

In one embodiment, in case of (f), the cell and the second adjuvant are administered only once in a time frame of from 0 h to 14 days after the administration of the delivery system of step i).

In a preferred embodiment, in case of (f), the cell and the second adjuvant are administered in a time frame of from 48 h to 14 days after the administration of the delivery system of step i).

For example, in case of (f), the cell and the second adjuvant are administered 48 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days after the administration of the delivery system of step i).

In a more preferred embodiment, in case of (f), the cell and the second adjuvant are administered in a time frame of from 3 days to 9 days, even more preferably of from 4 days to 8 days, for example 4 days, 5 days, 6 days, 7 days, 8 days after the administration of the delivery system of step i).

In another preferred embodiment, in case of (f), the cell and the second adjuvant are administered only once in a time frame of from 0 h to 14 days after the administration of the delivery system of step i).

For example, the cell and the second adjuvant are administered only once 48 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days after the administration of the delivery system of step i).

In a further preferred embodiment, in case of (f), the cell and the second adjuvant are administered only once in a time frame of from 3 days to 9 days, even more preferably of from 4 days to 8 days, for example 4 days, 5 days, 6 days, 7 days, 8 days after the administration of the delivery system of step i).

In case of (f), complexed interleukin 2 (IL-2cx) is preferably administered repeatedly after the administration of the delivery system of step i) in a time frame of from 0 h to 14 days after the administration of the delivery system of step i). It is further possible, that further administration of complexed IL-2 takes place after 14 days after the administration of the delivery system of step i); however, at least one administration is in a time frame of from 0 h to 14 days.

In a further preferred embodiment, in case of (f), complexed interleukin 2 (IL-2cx) is preferably administered in a time frame of from 48 h to 9 days, even more preferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the administration of the delivery system of step i).

In a yet further preferred embodiment, in case of (f), complexed interleukin 2 (IL-2cx) is preferably administered repeatedly in a time frame of from 48 h to 9 days, even more preferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the administration of the delivery system of step i). Thus, complexed interleukin 2 (IL-2cx) is preferably administered 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times in a time frame of from 48 h to 9 days, even more preferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the administration of the delivery system of step i). For example, complexed interleukin 2 (IL-2cx) may be administered daily or every two days. For example, complexed interleukin 2 (IL-2cx) may be administered 48 h, 3 days, 4 days, 5 days, 6 days, 7 days and 8 days after the administration of the delivery system of step i). In another example, complexed interleukin 2 (IL-2cx) may be administered 48 h, 4 days, 6 days, and 8 days after the administration of the delivery system of step i).

It is further possible, that further administration of complexed IL-2 takes place after the indicated time of, e.g. 14 days, preferably 9 or 8 days, after the administration of the delivery system of step i); however, at least one administration is in the indicated time frame.

Therefore, in a further preferred embodiment, in case of (f),

-   -   (A) the cell and the second adjuvant are administered in a time         frame of from 0 h to 14 days, preferably 48 h to 14 days, more         preferably in a time frame of from 3 days to 9 days, even more         preferably of from 4 days to 8 days, for example 4 days, 5 days,         6 days, 7 days, 8 days after the administration of the delivery         system of step i), even more preferably only once in a time         frame of from 48 h to 14 days, more particularly only once in a         time frame of from 3 days to 9 days, even more particularly only         once in a time frame of from 4 days to 8 days, for example 4         days, 5 days, 6 days, 7 days, 8 days after the administration of         the delivery system of step i), and     -   (B) complexed interleukin 2 (IL-2cx) is administered in a time         frame of from 0 h to 14 days, more preferably 48 h to 9 days,         even more preferably of from 3 days to 8 days, for example 3         days, 4 days, 5 days, 6 days, 7 days, 8 days after the         administration of the delivery system of step i), most         preferably administered repeatedly in a time frame of from 0 h         to 14 days, more preferably 48 h to 9 days, even more preferably         of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6         days, 7 days, 8 days after the administration of the delivery         system of step i).

It is further possible, that further administration of complexed IL-2 takes place after 14 days after the administration of the delivery system of step i); however, at least one administration is in a time frame of from 0 h to 14 days.

In a yet further preferred embodiment, in case of (f), complexed interleukin 2 (IL-2cx) is preferably administered repeatedly in a time frame of from 48 h to 9 days, even more preferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the administration of the delivery system of step i). Thus, complexed interleukin 2 (IL-2cx) is preferably administered 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times in a time frame of from 48 h to 9 days, even more preferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the administration of the delivery system of step i). For example, complexed interleukin 2 (IL-2cx) may be administered daily or every two days. For example, complexed interleukin 2 (IL-2cx) may be administered 48 h, 3 days, 4 days, 5 days, 6 days, 7 days and 8 days after the administration of the delivery system of step i). In another example, complexed interleukin 2 (IL-2cx) may be administered 48 h, 4 days, 6 days, and 8 days after the administration of the delivery system of step i).

It is further possible, that further administration of complexed IL-2 takes place after the indicated time of, e.g. 14 days, preferably 9 or 8 days, after the administration of the delivery system of step i); however, at least one administration is in the indicated time frame.

In another preferred embodiment, in case of (f), the cell and the second adjuvant are administered only once in a time frame of from 0 h to 14 days after the administration of the delivery system of step i).

The peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and the second adjuvant are preferably administered as a single pharmaceutical preparation. Such pharmaceutical preparation is preferably a liquid which may further contain pharmaceutical acceptable excipients like buffering compounds. Such pharmaceutical preparation is preferably sterilized.

In a preferred embodiment of the medicament for use,

-   -   (x) the complexed interleukin 2 (IL-2cx) is administered         repeatedly, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,         13 or 14 times, even more preferably wherein the complexed         interleukin 2 (IL-2cx) is administered every 1 or 2 days, and/or         is administered repeatedly during 5 days to 1 month, even more         preferably 1 to 2 weeks, and/or     -   (xx) the peptide derived from the antigen-comprising protein has         a length of 8, 9 or 10 amino acids and/or is a peptide presented         by a MHC-I, preferably by allele HLA-A2, HLA-A1, HLA-A3, HLA-B7,         HLA-B35, HLA-A24, or HLA-A30, more preferably by allele HLA-A2.

In a preferred embodiment, the complexed interleukin 2 (IL-2cx) is administered repeatedly, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 times, even more preferably wherein the complexed interleukin 2 (IL-2cx) is administered every 1 or 2 days, and/or is administered repeatedly during 5 days to 1 month. Therefore, it is possible, as described above, that further administration(s) of complexed IL-2 take place after the timeframe of 14 days, or 9 or 8 days.

As described above, a peptide-loaded major histocompatibility complex class I (MHC I) presenting cell and a second adjuvant are preferably administered as reactivator in step ii) of the medicaments for use of the present invention. Moreover, the peptide is derived from the antigen-comprising protein as defined in step i). “the peptide is derived from the antigen-comprising protein” is understood as that the peptide sequence is part of the antigen-comprising protein sequence (i.e. is a subsequence of antigen-comprising protein sequence).

A “peptide-loaded major histocompatibility complex class I (MHC I) presenting cell” is understood as cell which presents desired peptides on the surface of the cells by binding to MHC-I.

In one embodiment, the loading can be achieved by in vitro loading of MHC I molecules with peptides externally as described in the examples. Alternatively cells can be fed in vitro with whole antigen-comprising protein, allowing the cells to process the antigen and present the antigenic peptides in the context of MHC I, or they are exposed in vitro to viral systems capable of infecting the cells resulting in the expression of high amounts of a peptide in the context of MHC I, or the MHC I bearing cells could be transfected with expression vectors coding for a given protein or peptide sequence, again resulting in an efficient presentation of peptides in the context of MHC I.

Methods for determining a peptide for the loading of cells in the context of MHC-I are known in the art. For example, the methods described in Wu et al. (PNAS, 2011, 108(22): 9178-9183) may be used.

In case of applying defined peptides for loading, e.g. by externally loading cells in vitro, the peptide sequence may be chosen by methods known in the art. Externally loading cells in vitro can be performed by methods known in the art, e.g. by providing an aqueous solution of the peptides, adding the solution to the cells, which are preferably in a buffered solution or medium, incubating the cells with the peptides as to achieve a high saturation of the MHC-I and/or MHC-II with the respective peptide, and optionally washing the cells, e.g. with an aqueous solution.

In one preferred embodiment, cells are loaded in vitro with one peptide which has a sequence which is a subsequence of the antigen-comprising protein. For example, an aqueous solution comprising one such peptide may be added in vitro to a cell population, which is preferably a cell population of the patient.

Alternatively, a mixture of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different peptide loaded-cell major histocompatibility complex class I (MHC I) presenting cell populations may be used in step ii), wherein the cells are loaded with different peptides, and wherein the cells are preferably cells of the patient.

Such mixture of cells may be obtained by incubating a cell population, like PBMC cells, with a mixture of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different peptides, thereby obtaining cells loaded with different peptides in the context of MHC-I. Alternatively, separate cell populations, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cell populations, may be incubated in vitro with different peptides, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different peptides. Thereby, separate major histocompatibility complex class I (MHC I) presenting cell populations each loaded with a different peptide are thereby obtained. The different peptide loaded-cell major histocompatibility complex class I (MHC I) presenting cell populations may be administered separately in step ii), or a mixture of the different peptide loaded-cell major histocompatibility complex class I (MHC I) presenting cell populations may be prepared, which may then be administered in step ii).

In case different peptides are employed, in particular if 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different peptides are employed, such peptides may be derived from the same or a different antigen-comprising protein. In a preferred embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different peptides derived from one tumor antigen may be used. This means, that the sequence of each peptide is a subsequence of the tumor antigen. The sequences of such different peptides may be overlapping or non-overlapping. In a further embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different peptides derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different tumor antigen may be used. In such embodiment, the different tumor antigens are related to the same or different tumor, preferably to the same tumor.

Preferably, a peptide sequence of a length of 8, 9 or 10 amino acids is chosen, as peptides presented by MHC-I are typically of this length.

As further described in Wu et al., HLA alleles are extremely polymorphic. Therefore, the peptide loaded is preferably a peptide presented by a frequent HLA alleles. Therefore, in humans, a peptide presented the most frequent allele HLA-A2 is particularly preferred. Alternatively, peptides presented by HLA-A1, -A3, -B7, -B35, which are the alleles relevant for individuals of Caucasian origin may be used. HLA-A24 may be used for Asian individuals and HLA-A30 for African individuals.

Several suitable tumor-related peptides are described in Speiser and Romero (Seminars in Immunology 22 (2010) 144-154) and references therein. Most of them are HLA-A2 restricted, for example peptides derived from Melan-A/MART-1, one of the gp100 epitopes and tyrosinase for the melanocyte differentiation antigens; prostate surface antigen and PSAP for prostate; carcinoembryonic antigen and MUC-1 for mucosal tumors; HER-2/neu for breast carcinoma; G250 for renal cell carcinoma; the PR1 shared by two myeloid leukemia associated antigens, PR3 and neutrophil elastase which are normally expressed in granulocytes and overexpressed in myeloid leukemia cells; the shared tumor-specific antigens MAGE-A and NY-ESO-1 for various tumor types; and the overexpressed proteins survivin and telomerase. Suitable Influenza A-derived peptides are described in Wu et al (supra).

Therefore, in a further embodiment, the peptide derived from the antigen-comprising protein is a peptide presented by a MHC-I, preferably by allele HLA-A2, HLA-A1, HLA-A3, HLA-B7, HLA-B35, HLA-A24, or HLA-A30, more preferably by allele HLA-A2. Methods for identifying such peptides are described and summarized in Wu et al. (supra). For example, the systematic identification approach of Wu et al. (supra) may be used, or suitable algorithms described therein.

Therefore, in a further embodiment, the peptide derived from the antigen-comprising protein has a length of 8, 9 or 10 amino acids and is a peptide presented by a MHC-I, preferably by allele HLA-A2, HLA-A1, HLA-A3, HLA-B7, HLA-B35, HLA-A24, or HLA-A30, more preferably by allele HLA-A2.

In a further preferred embodiment of the medicament for use, the receptor on the surface of a dendritic cell is a receptor on the surface of cross-presenting dendritic cells.

Cross-presenting dendritic cells are particularly suitable for presenting peptides in the context of MHC-I. A cross-presenting DC is capable to take up soluble or targeted protein, process it, and present it in the context of MHC-I. All conventional DC are capable of antigen cross-presentation. Quantitatively optimal antigen cross-presentation is done by XCR1⁺ DC in the mouse and by XCR1⁺ DC within the BDCA3⁺ DC population in the human. Therefore, by XCR1⁺ DC are preferred murine cross-presenting DC, and XCR1⁺ DC within the BDCA3⁺ DC population are preferred human cross-presenting DC.

In a further preferred embodiment of the medicament for use, the receptor on the surface of a dendritic cell is chemokine (C motif) receptor 1 (XCR1), nectin-like molecule 2, a c-type lectin (CLEC) such as CLEC9A.

C-type lectins are Ca⁺⁺-dependent glycan-binding proteins that share primary and secondary structural homology in their carbohydrate-recognition domains (CRDs). These proteins have a C-type lectin fold, which is a fold with highly variable protein sequence that is also present in many proteins that do not bind carbohydrates.

The sequence of the human receptor CLEC9A is for example described in Caminschi et al., 2008, Blood 112, 3264-3273. A suitable molecule binding to CLEC9A is e.g. a specific monoclonal anti-CLEC9A antibody.

The human receptor nectin-like molecule 2 is described in Takai et al., 2003, Cancer Sci 94, 655-667 . . . A suitable molecule binding to nectin-like molecule 2 is e.g. a specific anti-nectin-like molecule 2 monoclonal antibody.

The delivery system is particularly suitable for influencing the Th1 response, and optionally also the Th2 response, in the immune system.

XCR1 is a chemokine receptor and is so far the only member of the “C” sub-family of chemokine receptors. It is also known as GPR5 or CCXCR1. GPR5, cloned previously as an orphan G-protein coupled receptor, has been recognized first in the human and then in the mouse as a monospecific receptor for XCL1 and was accordingly referred to as XCR1.

The natural ligand of XCR1 is XCL1, which is also known as ATAC, lymphotactin or SCM-1. It is the only member of the C family of chemokines. Activation-induced, T cell-derived, and chemokine-related cytokine (ATAC) was cloned in the human (Müller et al., 1995, Eur. J. Immunol. 25, 1744-48), and independently as lymphotactin (Kelner et al., 1994, Science 266, 1395-99) in the mouse and SCM-1 (Yoshida et al., 1995, FEBS Lett. 360, 155-9) in the human. According to the nomenclature on chemokines ATAC/lymphotactin/SCM-1 is now designated “XCL1”. XCL1 is secreted mainly by activated CD8⁺ T-cells, Th1 CD4⁺ T cells and by NK cells. In the human, a variant of XCL1 designated XCL2 has been described in which the amino acids aspartate and lysine in position 28 and 29 of the full length protein are exchanged for histidine and arginine, respectively (Yoshida et al., 1996, FEBS Lett. 395, 82-8), which may also be used for the present invention. An exemplary method to produce XCL1 in biologically active form is described in Example 8 of WO 2009/065561. Analogous methods may be used in order to produce other biologically active forms of XCL1, e.g. those of other species.

In an even more preferred embodiment, the receptor on the surface of a dendritic cell is XCR1.

The amino acid sequence of human XCR1 is known (NCBI; accession NP_001019815):

(SEQ ID NO: 12) MESSGNPEST TFFYYDLQSQ PCENQAWVFA TLATTVLYCL VFLLSLVGNS LVLWVLVKYE SLESLTNIFI LNLCLSDLVF ACLLPVWISP YHWGWVLGDF LCKLLNMIFS ISLYSSIFFL TIMTIHRYLS VVSPLSTLRV PTLRCRVLVT MAVWVASILS SILDTIFHKV LSSGCDYSEL TWYLTSVYQH NLFFLLSLGI ILFCYVEILR TLFRSRSKRR HRTVKLIFAI VVAYFLSWGP YNFTLFLQTL FRTQIIRSCE AKQQLEYALL ICRNLAFSHC CFNPVLYVFV GVKFRTHLKH VLRQFWFCRL QAPSPASIPH SPGAFAYEGA SFY

In a further preferred embodiment of the medicament for use, the molecule of a) is a ligand to the receptor or an antibody or antibody fragment against the receptor. In an even more preferred embodiment, the receptor is chemokine (C motif) receptor 1 (XCR1) and the molecule of a) is anti-XCR1 antibody or fragment thereof or chemokine (C motif) ligand 1 (XCL1) or a functionally active variant thereof, particularly comprising or consisting of the sequence of any of SEQ ID NOs: 7 to 10, preferably, of SEQ ID NOs: 8 to 10, more preferably of SEQ ID NOs: 9 or 10, especially of SEQ ID NO: 10.

The amino acid sequences of XCL1 (ATAC) of several species (including human: SEQ ID NO: 1, GenBank accession P47992; mouse: SEQ ID NO: 2, GenBank accession P47993; and rat SEQ ID NO: 3, GenBank accession P51672) are known and are shown as SEQ ID NO: 1 to 3 (see below). Additionally, a specific XCLR1 agonist referred to as K4.1 HHV8 (SEQ ID NO: 4, GenBank accession AAB62672.1) (see below), which is a viral chemokine-like protein, is also known. Any of these naturally occurring XCR1 ligands or any other natural occurring XCR1 ligand may be used.

Alternatively, a functionally active variant of any naturally occurring XCL1 may be used. The term variant encompasses fragments, variants derived by one or more amino acid additions, deletions and/or substitutions and molecules, particularly proteins, comprising any naturally occurring XCL1 or part thereof, such as fusion proteins. The XCL1 portion of the fusion protein may be flanked by the amino acid residue(s) C-terminally, N-terminally, or C- and N-terminally.

The functionally active fragment is characterized by being derived from any natural occurring XCR1 ligand, particularly XCL1, especially those of SEQ ID NO:1 to 4, by one or more amino acid deletions. The deletion(s) may be C-terminally, N-terminally and/or internally. Preferably, the fragment is obtained by at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50 or 60, more preferably by at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25 or 30, even more preferably at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, still more preferably at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, most preferably 1, 2, 3, 4 or 5 amino acid deletion(s). The functionally active fragment of the invention is characterized by having a biological activity similar to that displayed by the ligand from which it is derived, including the ability to binding to XCR1 and mediate internalization of a protein of (b). The fragment of the naturally occurring XCR1 ligand, particularly XCL1, especially those of SEQ ID NO:1 to 4, is functionally active in the context of the present invention, if the activity (binding as well as internalization) of the fragment amounts to at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 70%, still more preferably at least 80%, especially at least 90%, particularly at least 95%, most preferably at least 99% of the activity of the XCL1 without sequence alteration. These fragments may be designed or obtained in any desired length, including as small as about 18 to 50 amino acids in length.

The functionally active fragment of the naturally occurring XCR1 ligand, particularly XCL1, especially those of SEQ ID NO:1 to 4, may be also characterized by other structural features. Accordingly, in one preferred embodiment of the invention the functionally active fragments consists of at least 60%, preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95%, most preferably 99% of the amino acids of the XCR1 ligand of any of the SEQ ID NOS: 1 to 4. The functional active fragment as defined above may be derived from the peptide by one or more amino acid deletions. The deletions may be C-terminally, N-terminally and/or internally. The above sequence alignment of SEQ ID NOs: 1 to 4 shows domains of the naturally occurring ligands which seem to be conserved. In a preferred embodiment of the invention, these domains should be maintained in the fragment.

Conserved domains include those amino acids of the processed N-terminus (the processed N-terminus starting with amino acid 22 of non processed N-terminus) for SEQ ID NOs: 1 to 3 and with amino acid 27 for SEQ ID NO 4) at positions 1-2 (V/S G), 13-27 (S/N L X T/S Q/A R L P V/P X K/R I/L K/I X T/G X Y, X =any or no amino acid; SEQ ID NO: 5), 35 to 51 (R/K AV I F I/V T K/H R/S G L/R K/R I/V C A/G D/S P; SEQ ID NO: 6) and a disulfide bridge between cysteine residues at positions 11 and 48 (see also above alignment). A consensus sequence for the sequences of SEQ ID NO: 1 to 4 is XGXXXXXXXXXXCXXXLXXXRLPXXXXXXXXYXXXXXXXXXXAVIFXTXXGXX-XCXXP (SEQ ID NO: 7) if only identical amino acids are considered and (V/S)GX(E/A)(V/T)XXXXXXXC(V/E)X(S/N)LX(T/S)(Q/A)RLP(V/P)X(K/R)(I/L)(K/I)-X(T/G)XYX(I/T)X(E/T)(G/V)XXXX(R/K)AVIF(V/I)T(K/H)(R/S)G(L/R)(K/R)XC(A/G)-(D/S)P (SEQ ID NO: 8) if identical amino acids and majority amino acids (i.e. amino acids which are present in 3 of the 4 sequences, the alternative amino acid is listed after the slash) are considered. A consensus sequence for the sequences of SEQ ID NO: 1 to 3 is VGXEVXXXXXCVXLXTQRLPVXXIKTYXIXEGXXRA-VIFXTKRGLXXCADPXAXWVXXXXXXXDXXXXXXXXXXXTXPTXXQXSXXTAXT-LTG (SEQ ID NO: 9) if only identical amino acids are considered and VG(T/S)EV-(L/S)X(E/K)(S/R)XCV-(S/N)LXTQRLPV(Q/S)(K/R)IKTY(T/I)IXEG(A/S)(M/L)RAVIF(V/I)TKRGL(K/R)(I/V)-CADP(Q/E)A(K/T)VVV(K/R)X(A/V)(I/V)(K/R)(T/S)(V/M)D(G/R)(R/K)(A/S)(S/N)(T/A)-(R/S)(K/N)(N/S)(M/K)(AI) (E/Q)TXPT(G/Q)(A/T)Q(R/Q)S(T/A)(S/N)TA(V/I)TLTG (SEQ ID NO: 10) if identical amino acids and majority amino acids (i.e. amino acids which are present in 2 of the 3 sequences, the alternative amino acid is listed after the slash) are considered.

Accordingly, in a preferred delivery system for use in the invention the functionally active variant, preferably the functionally active fragment, of XCL1 comprises or consists of the sequence of any of SEQ ID NOs: 7 to 10, preferably of SEQ ID NOs: 8 to 10, more preferably of SEQ ID NOs: 9 or 10, especially of SEQ ID NO: 10.

In a further preferred embodiment of the invention, a XCL1 variant as defined above, wherein the XCR1 ligand is a functionally active variant of an XCR1 ligand of any of the SEQ ID NOS: 1 to 4 and wherein the variant has at least 50% sequence identity to the XCR1 ligand of any of the SEQ ID NOS: 1 to 4 is used. In a more preferred embodiment the functionally active variant has a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95%, most preferably 99% to the antigen of any of the SEQ ID NOS: 1 to 4.

The percentage of sequence identity can be determined e.g. by sequence alignment. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms have been described e.g. in Smith and Waterman, Adv. Appl. Math. 2: 482, 1981 or Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444-2448, 1988.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Variants of an antigen of any of the sequences of SEQ ID NOS: 1 to 4 are typically characterized using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of at least 35 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 35 amino acids), the alignment is performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Methods for determining sequence identity over such short windows such as 15 amino acids or less are described at the website that is maintained by the National Center for Biotechnology Information in Bethesda, Md. (http: //www.ncbi.nlm.nih.gov/BLAST/).

Alternatively, the alignment of multiple sequences may be performed using the MegAlign Sofware from DNAStar (Madison, Wis., USA) employing the ClustalV alignment algorithm (Higgins et al., 1992, Comput. Appl. Biosci. 8, 189-91). In the above alignment this software was used and set to the following default parameters: gap penalty 10, gap length penalty 10. Because of the very low homology, manual adjustments were necessary for the inclusion of SEQ ID NO 4 into the alignment.

The functional active variant is obtained by sequence alterations in the naturally occurring XCR1 ligand, wherein the XCR1 ligand with the sequence alterations retains a function of the unaltered XCR1 ligand, e.g. having a biological activity similar to that displayed by the naturally occurring XCR1 ligand, including the ability to binding to XCR1 and mediate internalization of a protein of (b) in step i). Such sequence alterations can include, but are not limited to, conservative substitutions, deletions, mutations and insertions. These characteristics of the functional active variant can be assessed e.g. as detailed above.

In a still more preferred embodiment the functionally active variant of XCL1 for use is derived from the naturally occurring XCR1 ligand of any of the sequences of SEQ ID NOS: 1 to 4 by conservative substitutions. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc. In one embodiment, one conservative substitution is included in the peptide. In another embodiment, two conservative substitutions or less are included in the peptide. In a further embodiment, three conservative substitutions or less are included in the peptide.

Examples of conservative amino acid substitutions include, but are not limited to, those listed below:

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Asn Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

A suitable monoclonal anti-XCR1 antibody for use according to the invention is for example mAb 6F8 disclosed in WO 2009/065561 or MARX10 (Bachem et al., 2012, Front Immunol 3, 214).

An anti-XCR1 antibody or functionally active fragment thereof may be used in one preferred embodiment as molecule binding to XCR1 as receptor in step i). An anti-XCR1 antibody or functionally active fragment thereof is capable of binding specifically to the XCR1. The functionally active fragment of the antibody is defined analogously to the functionally active fragment of XCL1 (see above), i.e. the functionally active fragment (a) is characterized by being derived from any anti-XCR1 antibody by one or more amino acid deletions, such as C-terminal, N-terminal and/or internal deletions and (b) is characterized by having a biological activity similar to that displayed by the anti-XCR1 antibody from which it is derived, including the ability to binding to XCL1. Naturally occurring antibodies are proteins used by the immune system to identify and neutralize foreign objects. Each naturally occurring antibody has two large heavy chains and two small light chains and can bind to a different antigen. The present invention includes, for example, monoclonal and polyclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, Fab, Fab′, F(ab′)2′, Fv, or the product of a Fab expression library. The antibody or antibody component can further be modified to prolong its biological half-life or in other ways to make them more suitable for targeting. Antibodies generated against XCR1 can be obtained by direct injection of XCR1 or a fragment thereof into an animal or by administering XCR1 or a fragment thereof to an animal, preferably a non-human. The antibody so obtained will then bind to XCR1. For preparation of monoclonal antibodies, any technique known in the art, which provides antibodies produced by continuous cell line cultures, e.g. a hybridoma cell line, can be used. The production of a suitable monoclonal antibody is also detailed in WO 2009/065561. Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to XCR1. Also, transgenic mice or other organisms such as other mammals may be used to express humanized antibodies to XCR1.

In a further preferred embodiment of the medicament for use,

-   -   the antigen-comprising protein of (b) is in a fusion protein         with the molecule of a); and/or     -   the antigen of the antigen-comprising protein of (b) is an         immunogen, a pathogen-derived antigen, or a tumor antigen.

Thus, in one preferred embodiment, the antigen-comprising protein of (b) is in a fusion protein with the molecule of a). Such fusion proteins can be synthesized e.g. synthetically or recombinantly, preferably recombinantly. Such fusion proteins enable efficient targeting to the dendritic cells. For example, fusion proteins of antibody MARX10 with OVA and XCL1 with OVA were successfully used in the Examples.

An immunogen is an antigen that stimulates an immune response. Antigens are substances recognized by specific receptors on T cells (T-cell receptor) and B cells (B-cell receptor) within the immune system and are usually proteins or polysaccharides. This includes parts (coats, capsules, cell walls, flagella, fimbrae, and toxins) of bacteria, viruses, and other microorganisms. In general, lipids and nucleic acids are antigenic only when combined with proteins and polysaccharides. Non-microbial exogenous (non-self) antigens can include pollen, egg white, and proteins from transplanted tissues and organs or on the surface of transfused blood cells.

Antigens can be categorized as endogenous or exogenous. A preferred antigen of the present invention is an exogenous antigen.

In case a pathogen-derived antigen is used, preferably of a virus, bacterium and/or eukaryotic parasite, the medicament is preferably for use in inducing a cellular cytotoxic immune response to an infection with such pathogen.

In case a tumor antigen is used, the medicament is preferably for use in inducing a cellular cytotoxic immune response to such tumor.

Dendritic cells can present exogenous antigens via MHC class I molecules, a process known as “cross-presentation”.

In a further preferred embodiment of the medicament for use, the first adjuvant of c) and second adjuvant are independently an adjuvant which supports a Th-1-mediated response, preferably they are independently selected from the group consisting of synthetic or recombinant RIG-I-agonists, TLR ligands, like poly ICLC and resiquimod (R848), Montanides, like ISA51, ISA720, saponins like Quil-A, ISCOM, QS-21, AS02 and AS01, polyinosinic:polycytidylic acid (poly I:C), a lipopolysaccharide (LPS), and a CpG oligodeoxynucleotide, more preferably selected from an RIG-I-agonist, and a TLR ligand, such as resiquimod (R848), poly ICLC and polyinosinic:polycytidylic acid (poly I:C).

If both a first adjuvant of c) and a second adjuvant are used, they may be the same or they may be different from each other.

Poly-ICLC consists of poly-IC stabilized with poly-L-lysine and carboxymethylcellulose and is potent in supporting a Th1 response.

R848 is a selective ligand for TLR7 in mice and for TLR7 and TLR8 in humans and activates the NLR pryin domain containing 3 (NLRP3) inflammasome.

For Poly-ICLC and R848, it is referred to Tacken and Figdor (supra) and references cited therein.

Montanides like ISA51 and ISA720 are water-in-oil emulsions containing squalene and mannide-monooleate as an emulsifier, as disclosed in Speiser and Romero and references cited therein.

Saponins like Quil-A, ISCOM, QS-21, AS02 and AS01 are triterpene glycosides isolated from plants, as disclosed in Speiser and Romero and references cited therein.

An adjuvant is an agent which modifies the effect of other agents while having few if any direct effects when given by itself. In pharmacology, adjuvants are drugs that have few or no pharmacological effects by themselves, but may increase the efficacy or potency of other drugs when given at the same time. In immunology an adjuvant is an agent which, while not having any specific antigenic effect in itself, may stimulate the immune system, increasing the response to a vaccine. The adjuvants used in the present invention are preferably supporting a Th1 response. An adjuvant supporting or inducing a Th1 response (or type 1 T cell response) is understood as adjuvant which is capable to induce IL-12 in DC, and leads to the secretion of IL-2, IFN-γ and TNF-α by responsive antigen-reactive CD8+ and CD4+ T cells, as well as production of the cytotoxic molecules granzyme B and perforin by antigen-reactive cytotoxic CD8+ and CD4+ T cells. Suitable preferred first and second adjuvants which support a Th1 response are known in the art and are for example described in Tacken and Figdor, and in Speiser and Romero. (Tacken P. J., Figdor C. G.; Targeted antigen delivery and activation of dendritic cells in vivo: Steps towards cost effective vaccines. Semin. Immunol. (2011), doi:10.1016/j.smim.2011.01.001; Speiser D. E. and Romero P., Seminars in Immunology 22 (2010) 144-154).

The dendritic cell (DC) is capable of sensing through a large set of “danger signal” recognition receptors (e.g. toll-like receptors, NOD-like receptors), whether the antigen is of dangerous nature or whether it is harmless. The patterns recognized by the “danger signal” recognition receptors (also designated “pattern recognition receptors”) are usually molecular structures that are unique to microorganisms. These can be cell wall components (e.g. lipopolysaccharide, peptidoglycan) or nucleic acid modifications (e.g. unmethylated CpG motifs) in case of microbes, or structural features and modifications that are unique to viral DNA or viral RNA (e.g. double-stranded RNA). Also cells dying from apoptosis in the body release molecules which are capable of triggering “danger signal” recognition receptors (e.g. High Mobility Group Protein B1, heat-shock proteins). Such danger signals are preferred first and second adjuvants of the present invention.

In a further preferred embodiment of the medicament for use, the re-activator is a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant, wherein the cell is a blood cell, especially a peripheral blood mononucleated cell (PBMC), preferably in combination with IL-2cx.

In a further preferred embodiment of the medicament for use, the T cell is a CD830 T cell or a CD4+ T cell, preferably a CD8+ T cell.

In particular, the re-activation of CD8+ T cells is specifically advantageous for obtaining a strong and enhanced cytotoxic immune response.

In a further preferred embodiment of the medicament for use, the time frame is from 72 h to 12 days, 72 h to 9 days, particularly from 5 days to 8 days, 5 days to 9 days or 5 days to 12 days.

In one preferred embodiment, the method steps i) and ii) are performed only once.

However, it is possible to repeat the method steps with the same delivery system and the same reactivator when the immune system has settled again. This is typically the case after at least one month, preferably after at least two months after inducing a cellular cytotoxic immune response according to the invention as described above.

Therefore, in another preferred embodiment, the method steps with the same delivery system according to step i) and the same reactivator according to step ii) is repeated after at least 1, 2, or 3 months after performing the method steps of the invention.

Alternatively, the method steps may be repeated with a different delivery system according to step i), preferably wherein the different delivery system comprises a different antigen-comprising protein (b) and optionally a different molecule binding to a receptor on the surface of a dendritic cell (a). In such embodiment, it is not necessary to wait until the immune system has settled again. Therefore, the method may therefore be repeated with a different delivery system e.g. 7 or 14 days after performing the method steps of the invention for the first time with a first delivery system. In such embodiment, the same or a different reactivator may be used in step ii).

In another embodiment, the present invention relates to a medicament for use in a method of extending a cellular cytotoxic immune response against an antigen-comprising protein, the method comprising the step of:

-   -   i) administering to a patient having T cells activated against         an antigen a peptide-loaded major histocompatibility complex         class I (MHC-I) presenting cell and a second adjuvant, wherein         the peptide is derived from the antigen-comprising protein,         thereby re-activating the activated T cell, and optionally         further administering complexed interleukin 2 (IL-2cx),

wherein the re-activator of step i) is administered in a time frame of from 0 h to 14 days after the T cells were activated against an antigen.

“Cellular cytotoxic immune response” is understood as Th1-type humoral and cellular (cytotoxic) immune reaction that can be elicited to a given antigen. This is in contrast to the response of classical vaccines which mainly address the Th2 antigen presentation pathway and mainly lead to the generation of Th2-type (neutralizing) antibodies and immune reactions.

An “Extended” cellular cytotoxic immune response is understood as a cellular cytotoxic immune response which occurs for a longer time compared to the cellular cytotoxic immune response obtained by a delivery system of step i) of a medicament for use of the present invention. For example, the response may be extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

For this embodiment, the same preferred embodiments apply, where applicable, as for above described medicaments for use of the present invention.

In an embodiment, the cell and the second adjuvant are administered in a time frame of from 0 h to 14 days after the T cells were activated against an antigen.

In one preferred embodiment, the cell and the second adjuvant are administered only once in a time frame of from 0 h to 14 days after the T cells were activated against an antigen.

In a further preferred embodiment, the cell and the second adjuvant are administered in a time frame of from 48 h to 14 days after the T cells were activated against an antigen.

For example, the cell and the second adjuvant are administered 48 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days after the T cells were activated against an antigen.

In a more preferred embodiment, the cell and the second adjuvant are administered in a time frame of from 5 days to 9 days, 5 days to 12 days or 3 days to 9 days, even more preferably of from 4 days to 8 days or 5 days to 12 days, for example 4 days, 5 days, 6 days, 7 days, 8 days after the T cells were activated against an antigen.

In another preferred embodiment, the cell and the second adjuvant are administered only once in a time frame of from 0 h to 14 days after the T cells were activated against an antigen.

In a further preferred embodiment, the cell and the second adjuvant are administered only once in a time frame of from 48 h to 14 days after the T cells were activated against an antigen.

For example, the cell and the second adjuvant are administered only once 48 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days after the T cells were activated against an antigen.

In a further preferred embodiment, the cell and the second adjuvant are administered only once in a time frame of from 5 days to 9 days, 5 days to 12 days or 3 days to 9 days, even more preferably of from 4 days to 8 days or 5 days to 12 days, for example 4 days, 5 days, 6 days, 7 days, 8 days after the T cells were activated against an antigen.

In one preferred embodiment, a combination of complexed interleukin 2 (IL-2cx), and a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant is administered. In a more preferred embodiment, the complexed interleukin 2 (IL-2cx) may be administered before, concomitantly or after administration of the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant. Preferably, the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant are only administered once, and the complexed interleukin 2 (IL-2cx) is administered repeatedly in a time frame of from 0 h to 14 days after the T cells were activated against an antigen. In such embodiment, complexed IL-2 may be administered both before, concomitantly and/or after administration of the peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant.

For of the combination, the same preferred embodiments for administration of peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant, apply as for the administration of the cells only.

In one embodiment, in case of the combination, the cell and the second adjuvant are administered only once in a time frame of from 0 h to 14 days after the T cells were activated against an antigen.

In a preferred embodiment, in case of the combination, the cell and the second adjuvant are administered in a time frame of from 48 h to 14 days after the T cells were activated against an antigen.

For example, in case of the combination, the cell and the second adjuvant are administered 48 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days after the T cells were activated against an antigen.

In a more preferred embodiment, in case of the combination, the cell and the second adjuvant are administered in a time frame of from 3 days to 9 days, even more preferably of from 4 days to 8 days, for example 4 days, 5 days, 6 days, 7 days, 8 days after the T cells were activated against an antigen.

In another preferred embodiment, in case of the combination, the cell and the second adjuvant are administered only once in a time frame of from 0 h to 14 days after the T cells were activated against an antigen.

For example, the cell and the second adjuvant are administered only once 48 h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days after the T cells were activated against an antigen.

In a further preferred embodiment, in case of the combination, the cell and the second adjuvant are administered only once in a time frame of from 3 days to 9 days, even more preferably of from 4 days to 8 days, for example 4 days, 5 days, 6 days, 7 days, 8 days after the T cells were activated against an antigen.

In case of the combination, complexed interleukin 2 (IL-2cx) is preferably administered repeatedly after the T cells were activated against an antigen in a time frame of from 0 h to 14 days after the T cells were activated against an antigen. It is further possible, that further administration of complexed IL-2 takes place after 14 days after the T cells were activated against an antigen; however, at least one administration is in a time frame of from 0 h to 14 days.

In a further preferred embodiment, in case of the combination, complexed interleukin 2 (IL-2cx) is preferably administered in a time frame of from 48 h to 9 days, even more preferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the T cells were activated against an antigen.

In a yet further preferred embodiment, in case of the combination, complexed interleukin 2 (IL-2cx) is preferably administered repeatedly in a time frame of from 48 h to 9 days, even more preferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the T cells were activated against an antigen. Thus, complexed interleukin 2 (IL-2cx) is preferably administered 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times in a time frame of from 48 h to 9 days, even more preferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the T cells were activated against an antigen. For example, complexed interleukin 2 (IL-2cx) may be administered daily or every two days. For example, complexed interleukin 2 (IL-2cx) may be administered 48 h, 3 days, 4 days, 5 days, 6 days, 7 days and 8 days after the T cells were activated against an antigen. In another example, complexed interleukin 2 (IL-2cx) may be administered 48 h, 4 days, 6 days, and 8 days after the T cells were activated against an antigen.

It is further possible, that further administration of complexed IL-2 takes place after the indicated time of, e.g. 14 days, preferably 9 or 8 days, after the administration of the delivery system of step i); however, at least one administration is in the indicated time frame.

Therefore, in a further preferred embodiment, in case of the combination,

-   -   (A) the cell and the second adjuvant are administered in a time         frame of from 0 h to 14 days, preferably 48 h to 14 days, more         preferably in a time frame of from 3 days to 9 days, even more         preferably of from 4 days to 8 days, for example 4 days, 5 days,         6 days, 7 days, 8 days after the T cells were activated against         an antigen, even more preferably only once in a time frame of         from 48 h to 14 days, more particularly only once in a time         frame of from 3 days to 9 days, even more particularly only once         in a time frame of from 4 days to 8 days, for example 4 days, 5         days, 6 days, 7 days, 8 days after the T cells were activated         against an antigen, and     -   (B) complexed interleukin 2 (IL-2cx) is administered in a time         frame of from 0 h to 14 days, more preferably 48 h to 9 days,         even more preferably of from 3 days to 8 days, for example 3         days, 4 days, 5 days, 6 days, 7 days, 8 days after the T cells         were activated against an antigen, most preferably administered         repeatedly in a time frame of from 0 h to 14 days, more         preferably 48 h to 9 days, even more preferably of from 3 days         to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8         days after the T cells were activated against an antigen.

It is further possible in case of the combination, that further administration of complexed IL-2 takes place after 14 days after the T cells were activated against an antigen; however, at least one administration is in a time frame of from 0 h to 14 days.

In a yet further preferred embodiment, in case of the combination, complexed interleukin 2 (IL-2cx) is preferably administered repeatedly in a time frame of from 48 h to 9 days, even more preferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the T cells were activated against an antigen. Thus, complexed interleukin 2 (IL-2cx) is preferably administered 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times in a time frame of from 48 h to 9 days, even more preferably of from 3 days to 8 days, for example 3 days, 4 days, 5 days, 6 days, 7 days, 8 days after the T cells were activated against an antigen. For example, complexed interleukin 2 (IL-2cx) may be administered daily or every two days. For example, complexed interleukin 2 (IL-2cx) may be administered 48 h, 3 days, 4 days, 5 days, 6 days, 7 days and 8 days after the T cells were activated against an antigen. In another example, complexed interleukin 2 (IL-2cx) may be administered 0 h, 48 h, 4 days, 6 days, and 8 days after the T cells were activated against an antigen.

It is further possible, that further administration of complexed IL-2 takes place after the indicated time of, e.g. 14 days, preferably 9 or 8 days, after the T cells were activated against an antigen; however, at least one administration is in the indicated time frame.

In another preferred embodiment, in case of the combination, the cell and the second adjuvant are administered only once in a time frame of from 0 h to 14, preferably 48 h to 14 days after the T cells were activated against an antigen.

Although we tested the effect of ADAS after targeting of antigen into XCR1⁺ DC, one can conclude from our results that any system leading to a significant initial activation (“priming”) or re-activation of CD8⁺ T cells and/or CD4⁺ T cells by T cell receptor triggering in vivo can be amplified with the ADAS approach. The T cell population could also be initially activated in vitro and later adoptively transferred in vivo, so that ADAS could then be used to amplify the T cell effector response in vivo.

Therefore, in one embodiment, T cells activated against an antigen can be obtained by performing a method as described above in a medicament for use in step i) using a delivery system.

In another preferred embodiment, T cells activated against an antigen can be obtained in vitro. To this end, unselected T cells, preferably obtained from the patient to be treated, are co-cultured with antigen or antigen-comprising protein and APC, and antigen-responsive T cells are selected, e.g. by an IFN-α secretion assay, and expanded with growth factors. Alternatively, antigen-specific T cells are sorted using appropriate tetramers or analogues thereof, exposed to antigen in the presence of APC and expanded with growth factors.

In a preferred embodiment of the medicament for use, the MHC-I presenting cell is a blood cell, especially a peripheral blood mononucleated cell (PBMC).

The MHC-I presenting cell is preferably a cell obtained from the patient to be treated. Methods for obtaining such cells are known to the skilled person. For example, blood may be retrieved and PBMC cells may then be isolated.

In a further preferred embodiment of the medicament for use, the complexed interleukin 2 (IL-2cx) is administered, preferably administered repeatedly, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 times, even more preferably wherein the complexed interleukin 2 (IL-2cx) is administered every 1 or 2 days, and/or is administered repeatedly during 5 days to 1 month, even more preferably 1 to 2 weeks.

In a further preferred embodiment of the medicament for use, the peptide derived from the antigen-comprising protein has a length of 8, 9 or 10 amino acids and/or is a peptide presented by a MHC-I, preferably by allele HLA-A2, HLA-A1, HLA-A3, HLA-B7, HLA-B35, HLA-A24, or HLA-A30, more preferably by allele HLA-A2.

In a further preferred embodiment of the medicament for use, the patient is a mammal, in particular a human.

In a further preferred embodiment of the medicament for use, the method of inducing a cellular cytotoxic immune response is for prophylactically treating or treating a tumor and/or an infection.

The medicament for use can be provided to protect from infection (“prophylactically treating”). Alternatively, such a medicament could be used for therapeutic purposes. The infected individual, which may not be able to mount a sufficient Th1 immune response to the pathogen, could be administered a medicament designed to induce and/or extend a cellular cytotoxic response, and would thus become capable of containing or eradicating the infection (“treating”). Examples would be malaria, tuberculosis, leishmania, prion diseases, orthomyxoviruses and in particular influenza, hepatitis A, hepatitis B, chronic hepatitis C, HIV and other lentiviruses, cytomegalovirus, herpes viruses, papillomaviruses, bunyaviruses, caliciviruses, filoviruses, flaviviruses and in particular hepatitis C virus, papillomaviruses, paramyxoviruses, a variety of respiratory viruses and other viruses, or any other infection specified in the description.

The vaccine could also be used to protect healthy individuals from developing tumors with known antigenic components (e.g. melanoma, prostate carcinoma) (“prophylactically treating a tumor”). Alternatively, medicament for use could be used to cure patients who already have developed tumors. Examples of such tumors would be human virus-induced tumors, in particular papillomavirus-induced tumors, HCV-induced tumors, hepatitis B-virus induced tumors and others viruses which induce tumors upon chronic infection. Moreover, suitable tumors to be treated are spontaneously arising solid tumors (e.g. melanoma, prostate cancer, breast cancer, adenocarcinoma of the gut, lung cancer) and leukemias.

A pathogen or infectious agent is a biological agent, especially a living microorganism, which causes disease or illness to its host. Pathogen, according to this invention, means preferably a virus, bacterium and/or eukaryotic parasite. A pathogen-derived antigen is an antigen derived from a pathogen.

Cross-presentation of antigen is also of central importance for the eradication of tumors in the body. Tumor cells and tumor antigens have to be taken up, processed, and presented by DC to elicit an anti-tumor immune response. Since the elimination of most tumors requires an effective cytotoxic Th1 T cell response, cross-presentation of tumor antigens is essential. Thus, for an effective anti-tumor response, cross-presenting DC play a pre-eminent role. As shown in Example 5, the medicaments for use of the present invention exhibit a surprising beneficial effect in a tumor model.

In one embodiment, the medicament for use comprises, preferably consists of, (a) a molecule binding to a receptor on the surface of a dendritic cell, and (b) an antigen-comprising protein bound to molecule of (a).

In another embodiment, the medicament for use comprises, preferably consists of, (a) a molecule binding to a receptor on the surface of a dendritic cell, (b) an antigen-comprising protein bound to molecule of (a) and (c) a first adjuvant.

In yet another embodiment, the medicament for use comprises, preferably consists of (A) (a) a molecule binding to a receptor on the surface of a dendritic cell, (b) an antigen-comprising protein bound to molecule of (a) and (c) a first adjuvant and (B) (d) complexed interleukin 2 (IL-2cx), (e) a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant, or (f) a combination of (d) and (e), wherein the peptide is derived from the antigen-comprising protein as defined in (A).

In another embodiment, the present invention relates to a kit-of-parts comprising, preferably consisting of, a delivery system as defined above and a re-activator as defined above.

In yet another embodiment, the present invention relates to a kit-of-parts comprising, preferably consisting of (A) complexed interleukin 2 (IL-2cx), and (B) a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant.

In a yet further embodiment, the present invention relates to a method of extending a cellular cytotoxic immune response against an antigen-comprising protein, the method comprising the step of:

-   -   ii) administering to a patient having T cells activated against         an antigen a peptide-loaded major histocompatibility complex         class I (MHC-I) presenting cell and a second adjuvant, wherein         the peptide is derived from the antigen-comprising protein,         thereby re-activating the activated T cell, and optionally         further administering complexed interleukin 2 (IL-2cx),

wherein the re-activator of step i) is administered in a time frame of from 0 h to 14 days after the T cells were activated against an antigen.

In a yet further embodiment, the present invention relates to method of inducing a cellular cytotoxic immune response, the method comprising the steps of:

-   -   i) administering to a patient a delivery system comprising (a) a         molecule binding to a receptor on the surface of a dendritic         cell, (b) an antigen-comprising protein bound to molecule of (a)         and (c) a first adjuvant, wherein upon binding of the molecule         of (a) to the receptor, the protein of (b) is internalized and         processed in the dendritic cell and the antigen comprised in the         protein is presented on the surface of the dendritic cell,         thereby activating a T cell in the patient; and         -   ii) administering to the patient a re-activator selected             from the group consisting of (d) complexed interleukin 2             (IL-2cx), (e) a peptide-loaded major histocompatibility             complex class I (MHC-I) presenting cell and a second             adjuvant, and (f) a combination of (d) and (e), wherein the             peptide is derived from the antigen-comprising protein as             defined in step i), thereby re-activating the T cell             activated in step i),

wherein the re-activator of step ii) is administered in a time frame of from 0 h to 14 days after the administration of the delivery system of step i).

For the methods of the present invention and the kits-of-parts of the present invention, the same embodiments apply as for the medicaments for use of the present invention.

FIGURES

FIG. 1 shows induction of cytotoxic activity after targeting of antigen into XCR1⁺ DC

FIG. 2 shows protection from infection or from seeding of cancer cells by the induced cytotoxic activity

FIG. 3 shows amplification of CD8⁺ T cell cytotoxicity obtained by injection of syngeneic lymphocytes loaded with antigenic peptide SIINFEKL (SEQ ID NO: 11)

FIG. 4 shows highly synergistic amplification of cytotoxic CD8⁺ T cells by co-application of peptide-loaded cells and complexed IL-2

FIG. 5 shows synergistic effects of antigen targeting and co-application of peptide-loaded cells and complexed IL-2 in the treatment of established tumors

FIG. 6 shows that the low frequency of cytotoxic CD8⁺ T cells after priming using various modes of vaccination can be strongly amplified with ADAS. (A) C57BL/6 animals were injected on day 0 with 200 μg of soluble, non-targeted ovalbumin (OVA), or with 5 μg of mAb MARX10-OVA, DEC-205-OVA, 33D1-OVA, MOPC21-OVA; in all cases, 10 μg poly I:C were co-injected as adjuvant. On day 5, blood samples were taken and the frequencies of SIINFEKL-specific CD8⁺ T cells determined by flow cytometry using a specific tetramer. (B) On day 5, the immune response to the OVA-derived peptide SIINFEKL was amplified with the ADAS procedure (injection of 10×10⁶ syngeneic splenocytes loaded with SIINFEKL together with 50 μg poly I:C as adjuvant). On day 10, the animals were sacrificed and the frequencies of SIINFEKL-specific CD8⁺ T cells were determined in the spleen using the tetramer. (C) C57BL/6 Batf3-KO animals were immunized as described in (A) and ADAS-treated, as described in (B), and the frequencies of SIINFEKL-specific CD8⁺ T cells determined in the spleen on day 10.

FIG. 7 (A) R9-SIINFEKL polypeptide does not externally bind to the MHC-I groove: C57BL/6 splenocytes were incubated at a density of 5×10⁶ cells/ml with SIINFEKL peptide at 1 μM for 2 h at 37° C., 5% CO₂ in complete RPMI1640 culture medium, or at a density of 2×10⁶ cells/ml with R9-SIINFEKL polypeptide at 1 μM for 4 h. Cells were washed twice, and stained with anti-SIINFEKL-H2K^(b) mAb (clone 25-D1.16) to determine the efficiency of peptide loading to the MHC-I groove. In addition, cells were co-stained with various lineage markers to identify different splenic cell populations and analyzed by flow cytometry. (B) R9-SIINFEKL can be used to transport antigen into the cytoplasmic compartment of primary cells and thus allow loading of MHC-I with derived peptides: C57BL/6 splenocytes were incubated at a density of 2×10⁶ cells with R9-SIINFEKL polypeptide at various concentrations (1-30 μM) for 7 h as above. Thereafter, the cells were washed twice, stained with mAb 25-D1.16 to determine the efficiency of peptide loading to the MHC-I groove, co-stained with lineage markers, and analyzed by flow cytometry. With 30 μM polypeptide a significant cell death was observed (not shown). (C) R9-SIINFEKL loaded primary cells can be used for ADAS: C57BL/6 mice were injected i.v. on day 0 with 10×10⁶ R9-SIINFEKL loaded (5 μM for 7 h) splenocytes, or for comparison with 10×10⁶ SIINFEKL-loaded (2 μM for 2 h) splenocytes, or with 2 μg MARX10-OVA (all with 10 μg poly I:C) for priming, and the frequency and cytotoxic potential (granzyme B, KLRG1, not shown) of CD8⁺ T cells were determined on day 5. Alternatively, C57BL/6 mice were primed with 2 μg MARX10-OVA and 10 μg poly I:C, and subjected to ADAS on day 5 by injection i.v. with SIINFEKL-loaded (2 μM for 2 h) splenocytes and 50 μg poly I:C (positive control). In parallel, C57BL/6 mice were primed i.v. with 2 μg MARX10-OVA, or 2 μg 33D1-OVA, or 2 μg 1D3-OVA, or 200 μg untargeted OVA (all together with 10 μg poly I:C) on day 0, and subjected to ADAS by injection i.v. of R9-SIINFEKL-loaded (5 μM for 7 h) syngeneic splenocytes and 50 μg poly I:C. The ADAS-induced expansion of SIINFEKL-specific CD8⁺ T cells was determined on day 10 by flow cytometry using a specific tetramer. All CD8⁺ T cells exhibited markers indicative of cytotoxicity (granzyme B, KLRG1, not shown).

FIG. 8 Administration of antigen can be dissociated from application of adjuvant in the priming step: (A) C57BL/6 mice were injected i.v. on day 0 with 2 μg MARX10-OVA together with 10 μg poly I:C as adjuvant, mixed in one solution. Alternatively, mice were injected on day -1 with 10 μg of poly I:C and on day 0 with 2 μg MARX10-OVA. Alternatively, mice were injected on day 0 with 2 μg MARX10-OVA and on day 1 with 10 μg poly I:C. In each experimental group, blood samples were taken on day 5 and the frequencies of SIINFEKL-specific CD8⁺ T cells determined by flow cytometry using a specific tetramer. (B) The mice described in (A) were subjected to the ADAS amplification procedure (i.v. injection of splenocytes externally loaded with SIINFEKL together with 50 μg of poly I:C) and the frequencies of SIINFEKL-specific CD8⁺ T cells in the spleen were determined by flow cytometry. Administration of antigen can be dissociated from application of adjuvant in the ADAS procedure.

FIG. 9 Highly synergistic amplification of cytotoxic CD8⁺ T cells by combining ADAS and administration of complexed IL-15: C57BL/6 animals were primed on day 0 with 2 μg MARX10-OVA and 10 μg poly I:C and analyzed for the total number of SIINFEKL-specific CD8⁺ T cells in the spleen on day 5 (prime) using a specific tetramer and flow cytometry. Some of the primed animals were on day 5 subjected to ADAS only (injection of 10×106 SIINFEKL-loaded splenocytes together with 50 μg poly I:C), some received ADAS and were injected i.p. with IL-2cx (as described in Examples 4 and 5) on days 6, 7, and 8, other mice received ADAS on day 5 and were injected i.p. with complexed IL-15 (IL-15cx) on day 6, 7, and 8. In all ADAS-treated groups, the total number of SIINFEKL-specific cytotoxic CD8⁺ T cells was determined on day 9. The dose of IL-15cx for 1 mouse was generated by incubating 2 pg IL-15 (Peprotech # 210-15) with 9.3 μg sIL-15R-Fc (R&D, #551-MR-100) at 37° C. for 20 min, PBS was added to 500 μl and the solution injected i.p.

FIG. 10 ADAS can amplify resting memory CD8⁺ T cells in an antigen-specific manner: C57BL/6 mice were on day -1 adoptively transferred with 2,000 or 10,000 of OT-I T cells and primed on day 0 with MARX10-OVA and 10 μg poly I:C. ADAS (injection of 10×10⁶ SIINFEKL-loaded splenocytes together with 50 μg poly I:C) was performed in all animals on day 5, and animals were analyzed for the frequency of (A) OT-I T cells and (B) endogenous, Thy 1.1-negative SIIFEKL-specific CD8⁺ T cells on days 10 and 40. Another group of mice was subjected to ADAS again on day 69, and analyzed for the frequency of (A) OT-I T cells and (B) endogenous SIINFEKL-specific CD8⁺ T cells on day 74.

FIG. 11 In vivo amplification of in vitro-activated antigen-specific CD8⁺ T cells by ADAS: Splenocytes were isolated from OT-I mice and cultured in complete medium with SIINFEKL peptide at 1.4 nM for 3 days. Thereafter, cells were washed with PBS and 5 ×10⁵ OT-I T cells (as determined by flow cytometry) were adoptively transferred into naïve C57BL/6 mice and the animals were treated by ADAS at various time points after transfer. Shown is the effect on the frequency of transferred CD8⁺ T cells when ADAS was performed on day 5 after adoptive transfer. The proportion of adoptively transferred CD8⁺ T cells of all CD8⁺ T cells was determined in the blood on day 9 using flow cytometry. The frequencies in the blood correspond in these experiments to the frequencies in the spleen of the animals.

EXAMPLES Example 1

Induction of Cytotoxic Activity After Targeting of Antigen into XCR1⁺ DC (FIG. 1)

The model antigen OVA was recombinantly fused to the XCR1-specific mAb MARX10 (Bachem, A. et al. Front Immunol 3 (2012) 214) or recombinantly fused to the chemokine ligand XCL1 (Hartung, E. et al. J Immunol 194 (2015) 1069-1079), which specifically binds to the XCR1-receptor. When MARX10-OVA or XCL1-OVA were injected i.v. into naïve C57BL/6 mice at low levels, the antigen was targeted into XCR1⁺ DC. If this antigen priming occurred in the presence of an adjuvant (3 μg LPS, CpG, or 10 μg poly I:C), substantial cytotoxic activity was induced (shown are data with poly I:C as adjuvant). This cytotoxic activity was tested by injecting the primed animals on day 6 i.v. with target cells (splenic lymphocytes), which were previously loaded with SIINFEKL (SEQ ID NO: 11) in vitro, an OVA-derived peptide. The target cells were labeled after loading with the fluorophore CFSE to a high degree, while non-loaded control splenic lymphocytes were labeled with CFSE to a low degree. As shown in FIG. 1A, priming animals with OVA+adjuvant resulted in cytotoxic activity which eliminated almost all SIINFEKL-loaded target cells. FIG. 1B shows a dose-response curve obtained with various amounts of antigen targeted to XCR1⁺ DC. Identical results were obtained after using MARX10-SIINFEKL or XCL1-SIINFEKL for targeting of the immunogenic peptide into XCR1⁺ DC.

Example 2

Protection from Infection or from Seeding of Cancer Cells by the Induced Cytotoxic Activity (FIG. 2)

C57BL/6 mice were primed with MARX10-OVA (containing 2 μg of OVA) and adjuvant (10 μg of poly I:C) or were left untreated. Five days later, all mice were infected with 1×10⁶ CFU (=5×LD₅₀) of a L. monocytogenes strain, into which the peptide sequence SIINFEKL (SEQ ID NO: 11) has been engineered recombinantly

(Foulds et al., 2002, J. Immunol. 168, 1528-1532). While all untreated mice died within 3 to 4 days, the induced level of cytotoxicity by antigenic priming fully protected all animals from disease (FIG. 2A).

C57BL/6 mice were primed with MARX10-OVA or XCL1-OVA (each containing 0.16 μg of OVA) and adjuvant (3 μg LPS) i.v., control animals were injected with PBS. Seven days later, all animals were injected with 5×10⁵ EG.7 cells, an aggressive syngeneic tumor line engineered to express OVA (Moore, M. W. et al. Cell 54 (1988) 777-785). While PBS-treated animals all exhibited strong tumor growth after 14 days, none of the immunized animals had any tumor tissue at the site of injection or elsewhere, indicating that the induced level of cytotoxicity protected the animals from tumor seeding (FIG. 2B).

Example 3

Amplification of CD8⁺ T Cell Cytotoxicity Obtained by Injection of Syngeneic Lymphocytes Loaded with Antigenic Peptide SIINFEKL (SEQ ID NO: 11) (FIG. 3)

C57BL/6 mice were primed with MARX10-OVA (containing 2 μg OVA) and an adjuvant (poly I, CpG, or LPS, shown are the data with 10 μg poly I:C) on days -20, -15, -10, -7, -5, or -3. On day 0, the primed animals were injected i.v. with 10×10⁶ splenocytes (FIG. 3A) which were loaded before injection with SIINFEKL (SEQ ID NO: 11) in vitro (“Antigen-Dependent Amplification System”, ADAS). Together with the peptide-loaded cells an adjuvant was injected (various amounts of LPS or poly I:C), shown are data with 50 μg of poly I:C. On day 10 animals were sacrificed and the total number of SIINFEKL-specific CD8⁺ T cells was determined in the spleen using SIINFEKL-specific tetramers and flow cytometry (FIG. 3A). The results demonstrated that the system used for amplification of antigen-specific CD8⁺ T cells was effective in a narrow timeframe between days 5 and 9 after the initial antigenic stimulation (FIG. 3A).

C57BL/6 mice were primed with MARX10-OVA (containing 2 μg OVA) and an adjuvant (10 μg poly I:C). Five days later, 10×10⁶ splenic lymphocytes, or purified T cells, dendritic cells, or B cells which were loaded before injection with SIINFEKL (SEQ ID NO: 11) in vitro were injected i.v. into the primed animals. Together with the peptide-loaded cells an adjuvant was injected (50 μg poly I:C). On day 10 after priming with MARX10-OVA, the animals were sacrificed and the total number of SIINFEKL-specific CD8⁺ T cells was determined in the spleen using SIINFEKL-specific tetramers and flow cytometry. The results showed that the amplification of the cytotoxic CD8⁺ T cell response can be achieved with various cell populations expressing MHC-I on the cell surface. The amplified cells expressed high levels of effector molecules (TNF-α, IFN-γ, granzyme B).

Example 4

Highly Synergistic Amplification of Cytotoxic CD8⁺ T Cells by Co-application of Peptide-loaded Cells and Complexed IL-2 (FIG. 4)

C57BL/6 mice were primed with MARX10-OVA (containing 2 μg OVA) and an adjuvant (10 μg poly I:C). One group of primed animals was injected on days 1,2, and 3 after priming with complexed IL-2 (IL-2cx) obtained by incubating IL-2 (2 μg) and the anti-IL-2 mAb JES6-5H4 (10 μg, (Sander, B. J Immunol Methods 166 (1993) 201-214) overnight at 4° C., and sacrificed on day 6 (“Primed+IL-2cx”). Another group of mice was injected on day 5 with SIINFEKL-loaded splenocytes (10×10⁶) and adjuvant (50 μg poly I:C) and sacrificed on day 10 (“Primed+ADAS”). Another group of mice was injected on day 5 with SIINFEKL-loaded splenocytes (10×10⁶) and adjuvant (50 μg poly I:C), and with IL-2cx on days 6, 7, 8, and 9, and sacrificed on day 10 (“Primed+ADAS+IL-2cx”). At the end of each experiment, the total number of SIINFEKL-specific CD8⁺ T cells was determined in the spleen using SIINFEKL-specific tetramers and flow cytometry. The experiment determined a highly synergistic effect of ADAS and IL-2cx in the amplification of antigen-specific cytotoxic CD8⁺ T cells.

Example 5

Synergistic Effects of Antigen Targeting and Co-application of Peptide-loaded Cells and Complexed IL-2 in the Treatment of Established Tumors (FIG. 5)

C57BL/6 mice were injected s.c. with 5×10⁵ EG.7 cells, an aggressive syngeneic tumor line engineered to express OVA (Moore, M. W. et al. Cell 54 (1988) 777-785). On day 6 after tumor injection mice were primed with MARX10-OVA (containing 2 μg OVA) and adjuvant (10 μg poly I:C), one group was left untreated. Six days after priming (day 11), one group of mice was injected with SIINFEKL-loaded splenocytes (10×10⁶) and adjuvant (50 μg poly I:C) (“Primed+ADAS”). Another primed group of mice was injected on days 12, 13, 14, 15, 16, 17, 18, 19, 21, and 23 with IL-2cx only (“Primed+IL-2cx”). Another primed group of mice was injected on day 11 with SIINFEKL-loaded splenocytes (10×10⁶) and adjuvant (50 μg poly I:C), and with IL-2cx on days 12, 13, 14, 15, 16, 17, 18, 19, 21, and 23 (“Primed+ADAS+IL-2cx”). On day 25 all mice were sacrificed (some control animals had to be sacrificed earlier, because the tumor became >1 cm in diameter). All data represent the mean average size of tumors (in mm²) in each treatment group (n=6). The results demonstrate that priming of the tumor-injected mice alone with targeted OVA was not effective. In contrast, either IL-2cx or ADAS alone were effective by strongly reducing the tumor mass for approximately 7-10 days in primed mice, but could not prevent the outgrowth of the tumor thereafter. When a combination of ADAS and IL-2cx was applied to primed animals, the tumor mass was strongly reduced and the tumor was fully controlled until the end of the experiment (some mice had fully rejected the tumor tissue).

Example 6

Various Modes of Immunization, Result in a Low-frequency of Primed Antigen-Specific CD8⁺ T Cells. These can be Generally Strongly Expanded and Differentiated to Killer CD8⁺ T Cells with the ADAS Procedure.

MAb MARX10 (Bachem et al., Front Immunol 3 (2012) 214, EP2641915A1) recognizes XCR1, the lineage marker for XCR1⁺ DC, Mab DEC-205 (NLDC-145, Kraal et al., 1986, obtained from Biolegend) recognizes the CD205 molecule expressed on murine XCR1⁺ DC, mAb 33D1 (Nussenzweig et al., PNAS 79 (1982) 161-165, obtained from ATCC) recognizes the DCIR2 molecule on SIRPa⁺ DC, mAb 1D3 recognizes CD19 on B cells (Krop et al. Eur J Immunol 26 (1996) 238-242, obtained from ATCC), mAb MOPC-21 (Potter et al., J Natl Cancer Inst 26 (1961) 1109-1137, obtained from Biolegend) does not recognize any molecule in the mouse and is therefore used as an IgG1 isotype control. XCL1 is the chemokine ligand for XCR1 and can be used for targeting of antigen to XCR1⁺ DC in the mouse and in the human (Hartung et al. J Immunol 194 (2015) 1069-1079.

The antigen-binding regions of the heavy and light chains of mAb DEC-205, 33D1, 1D3, MOPC-21 were identified by mass spectrometry and grafted onto the backbone of mAb DEC-205 by standard recombinant techniques, as described previously for mAb MARX10 (Hartung et al., J Immunol 194 (2015) 1069-1079). This backbone has been modified previously to minimize binding to Fc-receptors. All constructs were then modified in such a way as to accommodate OVA as a C-terminal fusion protein to each of the antibodies, as described previously for mAb MARX10 (Hartung et al., J Immunol 194 (2015) 1069-1079). XCL1-OVA was generated as described previously (Hartung et al., J Immunol 194 (2015) 1069-1079).

C57BL/6 animals were injected on day 0 with a high amount (200 μg) of soluble, non-targeted OVA, or with 5 μg of mAb MARX10-OVA, DEC-205-OVA, 33D1-OVA, MOPC21-OVA; in all cases, 10 μg poly I:C were co-injected as an adjuvant. On day 5, blood samples were taken and the frequencies of OVA-specific CD8⁺ T cells determined by flow cytometry using a H-2K^(b) tetramer loaded with SIINFEKL and capable to bind to SIINFEKL-specific CD8⁺ T cells. As shown in FIG. 6A, all modes of antigen application, either non-targeted, or targeted to XCR1⁺ DC, to SIRPα⁺ DC, or to B cells induced an initial expansion of SIINFEKL-specific CD8⁺ T cells (“priming”), resulting in a frequency of approximately 2-3% of all CD8⁺ T cells in the blood. On day 5, the immune response to the OVA-derived peptide SIINFEKL was amplified with the ADAS procedure (injection of 10×10⁶ SIINFEKL-loaded syngeneic splenocytes together with 50 μg poly I:C). On day 10, the animals were sacrificed and the frequency of SIINFEKL-specific CD8⁺ T cells determined in the spleen. In addition, several markers indicative of cytotoxicity were determined (KLRG1, perforin, granzyme B, data not shown). In all cases, the ADAS procedure amplified the initial frequency of SIINFEKL-specific CD8⁺ T cells approximately tenfold (FIG. 6B) and induced a phenotype indicative of cytotoxic T cells (not shown).

In parallel, Batf3-KO animals on the C57BL/6 background (animals which lack XCR1⁺ DC, Hildner et al. Science 322 (2008) 1097-1100) were primed with the non-targeted or targeted OVA reagents, as above. On day 5 the Batf3-KO animals were also treated by the ADAS procedure, as above. On day 10, all animal were sacrificed and the frequencies of SIINFEKL-specific CD8⁺ T cells were determined in the spleen. While priming, followed by ADAS gave high frequencies of SIINFEKL-specific CD8⁺ T cells in all C57BL/6 animals (FIG. 6B), no substantial SIINFEKL-specific response could be observed after ADAS in any of the Batf3-KO animals (FIG. 6C).

Several conclusions can be drawn from these experiments. Immunization using high levels of non-targeted protein, when applied together with a Th1 adjuvant, will result in an initial frequency of antigen-specific cytotoxic CD8⁺ T cells, as demonstrated by us and others previously (Hartung et al., J Immunol 194 (2015) 1069-1079). Targeting of antigen into DC makes this primary immunization much more effective, since only low amounts of antigen are required to achieve the same effect (Hartung et al, 2015, Caminschi et al. Front Immunol 3 (2012) 13). Surprisingly, even targeting of antigen to B cells (in this case via CD19, a surface molecule specifically expressed on B cells) was similarly effective to targeting of antigen to DC. This effect can either be explained by transfer of antigen from B cells to DC (Allan et al. Immunity 25 (2006) 153-162), or by “unspecific” binding of the targeting anybody to Fc-receptors on DC. The latter effect is most likely responsible for the efficiency of priming when using MOPC-21, an isotype control antibody which does not recognize any antigen in the mouse immune system. These results are fully compatible with earlier results, in which targeting of antigen to marginal metallophilic macrophages via the surface receptor Siglec-1 also led to the generation of low-frequency cytotoxic CD8⁺ T cells, but not in Batf3-KO animals (Backer et al. Proc Natl Acad Sci U S A 107 (2010) 216-221). Our experiments with Baf3-KO animals are in line with the general assumption that priming of naïve T cells has to occur by DC. In particular, these experiments indicate that XCR1⁺ DC are required for this initial CD8⁺ T cell priming. Thus, targeting of antigen into XCR1⁺ DC promises to be the most effective way of targeting protein antigens, nucleic acids coding for antigens, or viral systems coding for antigens in order to achieve a good primary CD8⁺ T cell response (“priming”).

Together, these results indicate that targeting of antigen using antibodies or targeting using receptor ligands (e.g. XCL1-OVA) to conventional DC, skin DC or other DC, such as monocyte-derived DC, pDC, to macrophages or other cells is far more efficient for induction of an initial cytotoxic response compared to the application of non-targeted antigen. It can be anticipated that all kinds of priming with protein antigens (for example, but no limited to, by employing liposomes, nanoparticles, and other systems as antigen carriers (Saroja et al. Int J Pharm Investig 1 (2011) 64-74) will give similar results, as long as the protein is applied in the context of a Th1 adjuvant. It is also well documented in the literature that a similarly low initial frequency of cytotoxic CD8⁺ T cells can be induced by non-protein immunization, such as, but not limited to, application or injection of DNA or DNA-based vaccines, or RNA or RNA-based vaccines, into the body using a variety of systems, either targeted or non-targeted (Saroja et al. Int J Pharm Investig 1 (2011) 64-74, Koup et al. Cold Spring Harb Perspect Med 1 (2011) a007252, Ulmer et al. Vaccine 30 (2012) 4414-4418, Kramps et al. Wiley Interdiscip Rev RNA 4 (2013) 737-749) Similar priming of CD8⁺ T cells can be achieved with viral systems or attenuated viruses (Draper et al. Nat Rev Microbiol 8 (2010) 62-73). Injection or application of RNA or DNA-based preparations or non-replicating viral systems or attenuated viruses does not necessarily require an additional Th1 adjuvant, since these agents are self-adjuvanted; i.e. these agents also represent Th1 aduvants themselves.

It is clear that in essentially all ways of vaccine delivery into the body, the initial CD8⁺ cytotoxic response will be relatively weak. Such a weak response will in many cases be insufficient to prevent infection, to treat an infection, or to eradicate cancerous tissue. Therefore, there is a need for a system which can strongly amplify the initial priming.

In our experiments, we demonstrate that in all cases in which the initial CD8⁺ cytotoxic response (priming) is insufficient to clear the infection or the tumor, it can be amplified using the ADAS procedure.

Also immunization of patients with tumor tissue or dead cells will lead to a priming of the CD8⁺ T cell compartment (de Gruijl et al. Cancer Immunol Immunother 57 (2008) 1569-1577) and thus will make these cells susceptible to the ADAS procedure.

Alternatively, primary CD8⁺ T cell activation can be achieved by isolating pre-existing tumor or pathogen-specific CD8⁺ T cells from a patient, activating and expanding them in vitro, and injecting them back into the patient. These adoptively transferred (re-injected) CD8⁺ T cells will be re-activated and further expanded and differentiated to cytotoxic T cells by the ADAS procedure.

The ADAS amplification can be in all cases be further augmented synergistically by subsequent injections of complexed IL-2 (Examples 4, 5) or complexed IL-15 (see Example 9).

In our experiments we used poly I:C as adjuvant. Similar results will be achieved with all type of Th1 adjuvants, such as, but not limited to, poly I:C, RIG-I agonists, and TLR8 agonists.

Example 7

Delivery of Antigen into the Cytoplasmic Compartment of Primary Cells for ADAS Leads to an Effective Loading of MHC-I with Antigen-derived Peptides

We have demonstrated that external loading of the MHC-I of a variety of primary cells (e.g. B cells, T cells, DC, splenocytes) with antigen-derived peptide (e.g. SIINFEKL) can be used for ADAS. We have anticipated that the same procedure will also work with a variety of methods introducing whole protein into primary cells or expressing a protein inside the cell, as described above.

To further illustrate this concept we have used a stretch of 9 arginine residues as a cell-penetrating peptide (CPP, Milletti 2012, Bechara et al., 2013) to transport an antigen-comprising protein (polypeptide) into primary cells. The entire sequence of this 37 aa-polypeptide (termed R9-SIINFEKL) is RRRRRRRRRGYPYDVPDYALEQLESIINFEKLTEWTS (SEQ ID No. 13).

R9-SIINFNEKL needs intracellular processing by the proteasome before a derived antigenic peptide (SIINFEKL) is presented on the cell surface in the context of MHC-I. Thus, the system can serve as a model for introducing a whole protein into a primary cell which is then processed by the proteasome into fragments, some of which are then presented on the cell surface in the context of MHC-I.

This polypeptide cannot bind directly, externally, into the groove of the MHC-I. To prove this point, we incubated splenocytes of C57BL/6 mice with the polypeptide R9-SIINFEKL at 1 μM for 4 h 37° C. in complete medium. In parallel, splenocytes were incubated with the peptide SIINFEKL, which can directly bind to MHC-I externally, at 1 μM for only 2 h. After incubation, the cells were washed and analyzed by flow cytometry using mAb 25-D1.16, which recognizes SIINFEKL in the context of MHC-I H2K^(b) (Porgador et al. Immunity 6 (1997) 715-726). While incubation of splenocytes with SIINFEKL gave a strong signal with macrophages, B cells, T cells, pDC, and DC, as expected (since SIINFEKL externally binds to MHC-I), no signal was obtained with the R9-SIINFEKL polypeptide (FIG. 7A). This experiment directly demonstrated that the R9-polypeptide cannot directly fit into the MHC-I groove and serve as antigen.

In the next step, splenocytes were incubated for 7 h at increasing concentrations (1-30 μM) of the R9-SIINFEKL polypeptide. After incubation and washing, the amount of SIINFEKL-loaded MHC-I was determined by staining with mAb 25-D1.16. As shown in FIG. 7B, all examined primary splenic cells exhibited a dose-dependent signal. Although not directly measured, it can be assumed that also the MHC-II was loaded in a similar manner. This experiment demonstrated that a polypeptide, once transported into a cytoplasmic compartment of a primary cell by a CPP, will be processed and presented in the context of the MHC-I (and MHC-II).

From this experiment, it can be deduced that the same procedure will also work with a whole protein, which has also to be processed before being presented in the context of the MHC-I (and MHC-II). In fact, a similar loading of the MHC-I with SIINFEKL has been demonstrated with whole OVA, to which a stretch of 9 arginine residues has been fused N-terminally fused using standard recombinant techniques. In that experiment, OVA was correctly processed and presented in the context of MHC-I, as determined by staining with mAb 25-D1.16 (Mitsui et al. J Invest Dermatol 126 (2006) 1804-1812). Thus, our experiment, which is in line with the literature, demonstrates that the introduction of non-processed polypeptides or proteins into the cell using the CPP principle will effectively load the MHC-I (and MHC-II) of this cell.

From this experiment, one can deduce that any type of internal loading of primary cells with proteins and unprocessed peptides will lead to it an effective MHC-I presentation of peptides derived from this material.

Such an internal loading of primary cells could similarly be achieved with other methods introducing peptides or proteins into cells, for example by, but not limited to, electroporation, or by introducing nucleic acids coding for a peptides and proteins by a variety of methods such as, but not limited to, transfection, lipofection, transduction, injection, ballistic injection, infection.

We then tested the biological potency of primary cells internally loaded using R9-SIINFEKL. To this end, splenocytes from C57BL/6 mice were incubated with R9-SIINFEKL at 5 μM for 7 hours, and washed. They were then injected on day 0 i.v. into naïve C57BL/6 animals and were compared to i.v. injection of SIINFEKL-loaded splenocytes, or to the i.v. injection of MARX10-OVA. All preparation were applied together with poly I:C as adjuvant. As can be seen in FIG. 7C, all methods of antigenic delivery induced a low frequency of SIINFEKL-specific cytotoxic CD8⁺ T cells, as assessed with a specific tetramer and by phenotypic analysis (expression of granzyme B, KLRG1), MARX10-OVA being the most efficient method of antigen delivery.

In the next step, the R9-SIINFEKL loaded splenocytes were assessed for their capacity in the ADAS procedure. In this experiment, priming by MARX10-OVA and ADAS with SIINFEKL-loaded splenocytes served as the positive control and gave around 20% of SIINFEKL-specific cytotoxic CD8⁺ T cells (FIG. 7C). R9-SIINFEKL-loaded splenocytes were similarly effective in the ADAS procedure after priming with MARX10-OVA, 33D1-OVA, 1D3-OVA or high amounts of non-targeted OVA as SIIFEKL-loaded splenocytes (compare also FIG. 7A). This experiment demonstrated that any type of effective internal loading of primary cells with antigen will be efficient for ADAS. This loading could be with unprocessed polypeptides, whole proteins, or with nucleic acids coding for peptides or proteins, or using infectious agents, or viral or bacterial vector systems recombinantly modified to encode a desired polypeptide or protein.

Example 8

Application of Th1 Adjuvant can be Dissociated in Time from Application of Antigen Both in the Priming Step and in the ADAS Procedure

It is currently generally assumed that antigen has to be applied together with an antigen in order to achieve immunization of the host. Therefore, antigen is usually mixed with the adjuvant and applied together. It is currently generally assumed that the adjuvant should ideally be even physically linked to the antigen to achieve optimal results. Therefore, it was very surprising for us to realize that a dissociation of antigen administration from adjuvant delivery in the priming and ADAS procedures leads to good and even better immune responses.

C57BL/6 mice were injected i.v. on day 0 with 2 μg MARX10-OVA together with 10 μg Poly I:C as adjuvant, mixed in one solution. Alternatively, mice were injected on day −1 with 10 μg of poly I:C and on day 0 with 2 μg MARX10-OVA. Alternatively, mice were injected on day 0 with 2 μg MARX10-OVA and on day 1 with 10 μg poly I:C. In each experimental group, blood samples were taken on day 5 and the frequency of SIINFEKL-specific CD8⁺ T cells determined by flow cytometry. As can be seen in FIG. 8A, joint administration of antigen and adjuvant on day 0 gave a priming frequency of around 2% of all CD8⁺ T cells. In contrast, application of adjuvant one day before antigen appeared ineffective. Very surprisingly, administration of antigen on day 0 and application of adjuvant on day 1 was most effective, giving a frequency of around 4% of all CD8⁺ T cells on day 5.

When all experimental groups were treated by the ADAS procedure (i.v. injection of splenocytes externally loaded with SIINFEKL and 50 μg of poly I:C), the group with the joint application of antigen and adjuvant had around 15% SIINFEKL-specific cytotoxic CD8⁺ T cells. Clearly the best result was achieved with the group in which adjuvant was applied 1 day after antigen (around 30% SIINFEKL-specific CD8⁺ T cells). Interestingly, even in the group in which poly I:C was applied 1 day before antigen, there was a low level of SIINFEKL-specific T cells (around 5%), indicating that a certain priming has been achieved even in this group (which then became measurable through the amplification achieved in the ADAS procedure).

These experiments clearly demonstrate that application of antigen and adjuvant can be dissociated in time in the priming procedure. In fact, the results indicate that application of adjuvant some time after administration of antigen is advantageous over a joint application of the components. Our results indicate that application of adjuvant even several days after application of antigen will be effective. The exact time frame cannot be determined in the human and has to be estimated also as 1-2, possibly up to 3 days.

Example 9

Highly Synergistic Amplification of Cytotoxic CD8⁺ T Cells by Combining ADAS and Administration of Complexed IL-15

We have demonstrated that injection of complexed IL-2 (IL-2cx =murine IL-2 complexed with an antibody blocking the binding of IL-2 to its high affinity receptor CD25) on days 1, 2, and 3 after priming with MARX10-OVA (2 μg) and poly I:C (10 μg) on day 0, did not substantially raise the number of primed CD8⁺ T cells on day 6. However, when the animals were primed with MARX10-OVA (2 μg) and poly I:C (10 μg) on day 0 and subjected to ADAS (injection of 10×10⁶ splenocytes loaded with SIINFEKL together with 50 μg poly I:C) on day 5, injection of IL-2cx on days 6, 7, 8, and 9 dramatically raised the number of SIINFEKL-specific CD8⁺ T cells in the spleen on day 10 (Example 4 and FIG. 4).

Without ADAS, injection of IL-2cx on days 6, 7, 8, and 9 did not substantially raise the number of SIINFEKL-specific CD8⁺ T cells in the spleen (not shown), but was effective in reducing a tumor burden. Obviously, the ADAS procedure reactivates the primed CD8⁺ T cells in such a manner that they become highly sensitive to the action of IL-2cx and this results in a strong expansion of cytotoxic CD8⁺ T cells. However, the exact molecular mechanism leading to this highly heightened sensitivity to IL-2cx remains undetermined.

C57BL/6 animals were primed on day 0 with 2 μg MARX10-OVA and 10 μg poly I:C and subjected to ADAS (injection of 10×10⁶ SIINFEKL-loaded splenocytes together with 50 μg poly I:C) on day 5. As described earlier, the ADAS procedure raised the level of SIINFEKL-specific CD8⁺ T cells from around 200,000 on day 5 to around 2×10⁶ cells after ADAS on day 10 (FIG. 9). ADAS-treated animals were then injected on days 6, 7, and 8 with complexed IL-15 (IL-15cx), or for comparison with IL-2cx (as described above). On day 9, the total number of SIINFEKL-specific CD8⁺ T cells was determined in the spleen using flow cytometry. As can be seen in FIG. 9, repeated injection of IL-15cx was similarly effective to the repeated injection of IL-2cx in strongly amplifying the number of SIINFEKL-specific CD8⁺ T cells after the ADAS procedure. IL-15cx could be injected, like IL-2cx, i.v., s.c., i.p., or into a tumor to achieve this effect. Injection of IL-15cx also increased the cellular levels of granzyme B and expression of KLRG1, indicating augmented cytotoxicity (not shown).

Surprisingly, this experiment thus revealed that IL-15cx could strongly amplify the levels of ADAS-reactivated antigen-specific CD8⁺ T cells, similar to the action of IL-2cx. From this experiment it can be deduced that in all cases in which injection of IL-2cx was beneficial for the augmentation of the cytotoxic immune response (as described above) also IL-15cx will be effective.

Example 10

The ADAS Procedure can be Used to Re-activate and Expand Memory CD8⁺ T Cells in an Antigen-specific Manner

With naïve animals, which were primed with 2 μg MARX10-OVA and 10 μg poly I:C on day 0, we could demonstrate that ADAS is rather ineffective for an amplification of the response when performed on day 3, but becomes effective thereafter, and continues to be effective to a certain extent at least until day 20 (Example 3 and FIG. 3), and is most effective between days 4 and 9 in mice.

In these experiments, ADAS was used to amplify freshly activated naïve antigen-specific CD8⁺ T cells. The fact that ADAS only optimally works in a certain time window following primary activation of CD8⁺ T cells indicated that these T cells must be in a particular activation stage in order to be sensitive for the ADAS amplification.

We therefore wondered whether ADAS could also be performed on resting memory CD8⁺ T cells, which have a clearly different activation status compared to freshly activated CD8⁺ T cells. To this end, C57BL/6 mice were on day −1 adoptively transferred with 2,000 or 10,000 of OT-I CD8⁺ T cells, which bear a T cell receptor specific for SIINFEKL presented in the context of H-2K^(b). These transferred OT-I T cells bore the genetic marker Thy1.1 (CD90.1), which made it possible to discriminate them from endogenous T cells (compare Dorner et al., 2009). On day 0, the animals were primed with MARX10-OVA together with 10 μg poly I:C. ADAS (injection of 10×10⁶ SIINFEKL-loaded splenocytes together with 50 μg poly I:C) was performed on day 5, resulting in a frequency of 30% of OT-I T cells of all CD8⁺ T cells in the spleen on day 10 for both groups of adoptively transferred animals (FIG. 10A). When animals from the same experimental groups were analyzed on day 40, the frequency of their OT-I CD8⁺ T cells had declined to 1-2%, as expected (FIG. 10A). On day 69, another ADAS amplification was performed (injection of 10×10⁶ splenocytes loaded with SIINFEKL together with 50 μg poly I:C) and the frequency of OT-I T cells was determined in the spleen 5 days later (day 74). Through the ADAS amplification, the frequency of OT-I T cells again rose to 30-60% of all CD8⁺ T cells in the spleen.

In all animals adoptively transferred with OT-I T cells, we also analyzed the response of endogenous CD8⁺ T cells, since these could be identified as CD8⁺ T cells negative for the Thy 1.1 marker, but staining with the SIINFEKL-tetramer. As can be seen in FIG. 10B, the endogenous, SIINFEKL-reactive CD8⁺ T cells showed a behaviour similar to the OT-I T cells. In particular, the ADAS procedure could highly amplify endogenous SIINFEKL-specific memory T cells and these T cells bore all phenotypic markers typical of cytotoxic CD8⁺ T cells (granzyme B positivity, KLRG1-expression, data not shown).

These experiments determined that the ADAS procedure can reactivate and massively expand resting memory CD8⁺ T cells in an antigen-specific manner, so that they again become CD8⁺ cytotoxic effectors. Since the ADAS procedure generates a state of T cells in which they are responsive to IL-2cx and IL-15cx, both cytokine preparations will further strongly augment the response of memory CD8⁺ T cells to ADAS.

In summary, ADAS can not only amplify freshly activated naïve CD8⁺ T cells in an antigen-specific manner in a certain time window after activation, but also resting memory CD8⁺ T cells.

Example 11

ADAS is also Effective with In Vitro Activated, Adoptively Transferred CD8+ T Cells

In adoptive T cell therapy, antigen-specific CD8⁺ T cells (e.g. T cells directed to CMV antigens or any other pathogenic antigens or T cells directed to defined tumor antigens) are enriched from the PBMC of a patient. This is done, for example, using IFN-γ secretion capture after in vitro stimulation of the PBMC with an antigenic peptide or protein from this pathogen (e.g. CMV) or tumor, combined with magnetic cell sorting. The enriched antigen-specific CD8⁺ T cells are then further stimulated by addition of whole antigen or, more often, peptide antigen, and then expanded in vitro. After expansion, the activated CD8⁺ T cells are then re-injected into the patient in order to achieve the desired therapeutic effect (elimination of the pathogen or tumor). Currently, the re-injected CD8⁺ T cells have a short life span in vivo, their capacity to secrete IFN-γ is limited, and their cytotoxic potential is suboptimal. We have found that this shortcoming of the adoptive T cell therapy can be very substantially improved using the ADAS procedure.

Splenocytes of OT-I mice were in vitro activated with the peptide SIINFEKL for 3 days, without adjuvant. This activation of CD8⁺ T cells could, however, also be done in the presence of a Th1 adjuvant. After culture, the cells (at this time composed of 97% OT-I CD8⁺ T cells) were transferred into syngeneic C57BL/6 mice on day 0. ADAS (injection of 10×10⁶ SIIFEKL-loaded splenocytes and 50 μg poly I:C i.v.) was applied on either day 0, 1, 2, 3, 4, 5, or 6. ADAS on days 1, 2 or 3 did not have any significant effect on the frequency of the adoptively transferred CD8⁺ T cells in the host animals. Clear amplification of the transferred CD8⁺ T cells could, however, be observed with ADAS applied on day 4, and optimal amplification was seen with ADAS on day 5 (FIG. 11). At the same time, the expression of the surface marker KLRG1, indicative of maturation of the transferred CD8⁺ T cells to effector cells, rose from 30-40% with ADAS on day 1 to 80-90% on days 5 and 6. These data allow concluding that the ADAS procedure is capable to strongly amplify a population of in vitro-activated and adoptively transferred antigen-specific CD8⁺ T cells. At the same time, ADAS induces a further differentiation of these CD8⁺ T cells to cytotoxic effector cells. The optimal time point for ADAS may differ in the human, since experiments in the mouse do not allow a precise prediction in the human. Thus, the period for optimal ADAS effects on the adoptively transferred CD8⁺ T cells may be more extended for example until 12 or 14 days. It can also be anticipated that ADAS is not only effective within the short time frame after adoptive transfer of from 0 h to 14 days, but also when the adoptively transferred CD8⁺ T cells have returned to the memory state after a prolonged period of time. 

1. A medicament for use in a method of inducing a cellular cytotoxic immune response, the method comprising the steps of: i) administering to a patient a delivery system comprising (a) a molecule binding to a receptor on the surface of a dendritic cell, (b) an antigen-comprising protein bound to molecule of (a) and (c) a first adjuvant, wherein upon binding of the molecule of (a) to the receptor, the protein of (b) is internalized and processed in the dendritic cell and the antigen comprised in the protein is presented on the surface of the dendritic cell, thereby activating a T cell in the patient; and ii) administering to the patient a re-activator selected from the group consisting of (d) complexed interleukin 2 (IL-2cx), (e) a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant, and (f) a combination of (d) and (e), wherein the peptide is derived from the antigen-comprising protein as defined in step i), thereby re-activating the T cell activated in step i), wherein the re-activator of step ii) is administered in a time frame of from 0 h to 14 days after the administration of the delivery system of step i).
 2. The medicament for use of claim 1, wherein (x) the complexed interleukin 2 (IL-2cx) is administered repeatedly, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 times, even more preferably wherein the complexed interleukin 2 (IL-2cx) is administered every 1 or 2 days, and/or is administered repeatedly during 5 days to 1 month, even more preferably 1 to 2 weeks, and/or (xx) the peptide derived from the antigen-comprising protein has a length of 8, 9 or 10 amino acids and/or is a peptide presented by a MHC-I, preferably by allele HLA-A2, HLA-A1, HLA-A3, HLA-B7, HLA-B35, HLA-A24, or HLA-A30, more preferably by allele HLA-A2.
 3. The medicament for use of claim 1 or 2, wherein the receptor on the surface of a dendritic cell is a receptor on the surface of cross-presenting dendritic cells.
 4. The medicament for use of claim 2 or 3, wherein the receptor on the surface of a dendritic cell is chemokine (C motif) receptor 1 (XCR1), nectin-like molecule 2, a c-type lectin (CLEC) such as CLEC9A, preferably the receptor on the surface of a dendritic cell is XCR1.
 5. The medicament for use of any of claims 1 to 4, wherein the molecule of a) is a ligand to the receptor or an antibody or antibody fragment against the receptor, particularly wherein the receptor is chemokine (C motif) receptor 1 (XCR1) and wherein the molecule of a) is anti-XCR1 antibody or fragment thereof or chemokine (C motif) ligand 1 (XCL1) or a functionally active variant thereof, particularly comprising or consisting of the sequence of any of SEQ ID NOs: 7 to 10, preferably, of SEQ ID NOs: 8 to 10, more preferably of SEQ ID NOs: 9 or 10, especially of SEQ ID NO:
 10. 6. The medicament for use of any of claims 1 to 5, wherein the antigen-comprising protein of (b) is in a fusion protein with the molecule of a); and/or the antigen of antigen-comprising protein of (b) is an immunogen, a pathogen-derived antigen, or a tumor antigen.
 7. The medicament for use of any of claims 1 to 6, wherein the first adjuvant of c) and second adjuvant are independently an adjuvant which supports a Th-1-mediated response, preferably they are independently selected from the group consisting of synthetic or recombinant RIG-I agonists, TLR ligands, such as resiquimod (R848), poly ICLC or polyinosinic:polycytidylic acid (poly I:C) Montanides, saponins, a lipopolysaccharide (LPS), and a CpG oligodeoxynucleotide, more preferably selected from an RIG-I-agonist, and a TLR ligand, such as resiquimod (R848), poly ICLC or polyinosinic:polycytidylic acid (poly I:C).
 8. The medicament for use of any of claims 1 to 7, wherein the re-activator is a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant, wherein the cell is preferably a blood cell, especially a peripheral blood mononucleated cell (PBMC), more preferably in combination with IL-2cx, and/or the cell and the second adjuvant are administered only once in a time frame of from 0 h to 14 days after the administration of the delivery system of step i).
 9. The medicament for use of any of claims 1 to 8, wherein the T cell is a CD8+ T cell or a CD4+ T cell, preferably a CD8+ T cell.
 10. The medicament for use of any of claims 1 to 9, wherein the time frame is from 72 h to 12 days, preferably 72 h to 9 days, more preferably from 5 days to 8 days, 5 days to 9 days or 5 days to 12 days.
 11. A medicament for use in a method of extending a cellular cytotoxic immune response against an antigen-comprising protein, the method comprising the step of: i) administering to a patient having T cells activated against an antigen a peptide-loaded major histocompatibility complex class I (MHC-I) presenting cell and a second adjuvant, wherein the peptide is derived from the antigen-comprising protein, thereby re-activating the activated T cell, and optionally further administering complexed interleukin 2 (IL-2cx), wherein the re-activator of step i) is administered in a time frame of from 0 h to 14 days after the T cells were activated against an antigen, preferably wherein the re-activator of step i) is administered only once in a time frame of from 0 h to 14 days after the T cells were activated against an antigen.
 12. The medicament for use of claim 11, wherein (x) the MHC-I presenting cell is a blood cell, especially a peripheral blood mononucleated cell (PBMC), and/or (xi) the complexed interleukin 2 (IL-2cx) is administered, preferably administered repeatedly, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 times, even more preferably wherein the complexed interleukin 2 (IL-2cx) is administered every 1 or 2 days, and/or is administered repeatedly during 5 days to 1 month, even more preferably 1 to 2 weeks, and/or (xii) the peptide derived from the antigen-comprising protein has a length of 8, 9 or 10 amino acids and/or is a peptide presented by a MHC-I, preferably by allele HLA-A2, HLA-A1, HLA-A3, HLA-B7, HLA-B35, HLA-A24, or HLA-A30, more preferably by allele HLA-A2.
 13. The medicament for use of any of claims 1 to 12, wherein the patient is a human.
 14. The medicament for use of any of claims 1 to 13, wherein the method of inducing a cellular cytotoxic immune response is for prophylactically treating or treating a tumor and/or an infection.
 15. Kit-of-parts comprising a delivery system as defined in any of claims 1 and 5 and a re-activator as defined in claim 1 or
 8. 